Nucleic acid molecules encoding osteogenic proteins

ABSTRACT

Disclosed are 1) osteogenic devices comprising a matrix containing osteogenic protein and methods of inducing endochondral bone growth in mammals using the devices; 2) amino acid sequence data, amino acid composition, solubility properties, structural features, homologies and various other data characterizing osteogenic proteins, 3) methods of producing osteogenic proteins using recombinant DNA technology, and 4) osteogenically and chondrogenically active synthetic protein constructs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.09/754,831, filed Jan. 3, 2001, now U.S. Pat. No. 7,078,221, which is acontinuation of U.S. Ser. No. 08/375,901, filed Jan. 20, 1995, now U.S.Pat. No. 6,261,835, which is a divisional of U.S. Ser. No. 08/145,812,filed Nov. 1, 1993, now U.S. Pat. No. 5,750,651, which is a divisionalof U.S. Ser. No. 07/995,345, filed Dec. 22, 1992, now U.S. Pat. No.5,258,494, which is a divisional of U.S. Ser. No. 07/315,342, filed Feb.23, 1989, now U.S. Pat. No. 5,011,691, the disclosures of all of whichare incorporated herein by reference. This application—also relates toU.S. Ser. No. 07/232,630, entitled “Osteogenic Devices”, filed Aug. 15,1988, now abandoned, and U.S. Ser. No. 07/179,406, entitled “OsteogenicDevices”, filed Apr. 8, 1988, now U.S. Pat. No. 4,968,590, thedisclosures of both of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

This invention relates to osteogenic devices, to genes encoding proteinswhich can induce osteogenesis in mammals and methods for theirproduction using recombinant DNA techniques, to synthetic forms ofosteogenic protein, to a method of reproducibly purifying osteogenicprotein from mammalian bone, to matrix materials which act as a carrierto induce osteogenesis in mammals, and to bone and cartilage repairprocedures using the osteogenic device.

Mammalian bone tissue is known to contain one or more proteinaceousmaterials, presumably active during growth and natural bone healing,which can induce a developmental cascade of cellular events resulting inendochondral bone formation. This active factor (or factors) hasvariously been referred to in the literature as bone morphogenetic ormorphogenic protein, bone inductive protein, osteogenic protein,osteogenin, or osteoinductive protein.

The developmental cascade of bone differentiation consists of chemotaxisof mesenchymal cells, proliferation of progenitor cells, differentiationof cartilage, vascular invasion, bone formation, remodeling, and finallymarrow differentiation (Reddi (1981) Collagen Rel. Res. 1: 209-226).

Though the precise mechanisms underlying these phenotypictransformations are unclear, it has been shown that the naturalendochondral bone differentiation activity of bone matrix can bedissociatively extracted and reconstituted with inactive residualcollagenous matrix to restore full bone induction activity (Sampath andReddi, (1981) Proc. Natl. Acad. Sci. USA 21: 7599-7603). This providesan experimental method for assaying protein extracts for their abilityto induce endochondral bone in vivo.

This putative bone inductive protein has been shown to have a molecularmass of less than 50 kilodaltons (kD). Several species of mammalsproduce closely related protein as demonstrated by cross species implantexperiments (Sampath and Reddi (1983) Proc. Natl. Acad. Sci. USA 80:6591-6595).

The potential utility of these proteins has been widely recognized. Itis contemplated that the availability of the pure protein wouldrevolutionize orthopedic medicine, certain types of plastic surgery, andvarious periodontal and craniofacial reconstructive procedures.

The observed properties of these protein fractions have induced anintense research effort in various laboratories directed to isolatingand identifying the pure factor or factors responsible for osteogenicactivity. The current state of the art of purification of osteogenicprotein from mammalian bone is disclosed by Sampath et al. (Proc. Natl.Acad. Sci. USA (1987) 80). Urist et al. (Proc. Soc. Exp. Biol. Med.(1984) 17: 194-199) disclose a human osteogenic protein fraction whichwas extracted from demineralized cortical bone by means of a calciumchloride-urea inorganic-organic solvent mixture, and retrieved bydifferential precipitation in guanidine-hydrochloride and preparativegel electrophoresis. The authors report that the protein fraction has anamino acid composition of an acidic polypeptide and a molecular weightin a range of 17-18 kD.

Urist et al. (Proc. Natl. Acad. Sci. USA (1984) 11: 371-375) disclose abovine bone morphogenetic protein extract having the properties of anacidic polypeptide and a molecular weight of approximately 18 kD. Theauthors reported that the protein was present in a fraction separated byhydroxyapatite chromatography, and that it induced bone formation inmouse hindquarter muscle and bone regeneration in trephine defects inrat and dog skulls. Their method of obtaining the extract from boneresults in ill-defined and impure preparations.

European Patent Application Serial No. 148,155, published Oct. 7, 1985,purports to disclose osteogenic proteins derived from bovine, porcine,and human origin. One of the proteins, designated by the inventors as aP3 protein having a molecular weight of 22-24 kD, is said to have beenpurified to an essentially homogeneous state. This material is reportedto induce bone formation when implanted into animals.

International Application No. PCT/087/01537, published Jan. 14, 1988,discloses an impure fraction from bovine bone which has bone inductionqualities. The named applicants also disclose putative bone inductivefactors produced by recombinant DNA techniques. Four DNA sequences wereretrieved from human or bovine genomic or cDNA libraries and apparentlyexpressed in recombinant host cells. While the applicants stated thatthe expressed proteins may be bone morphogenic proteins, bone inductionwas not demonstrated, suggesting that the recombinant proteins are notosteogenic. See also Urist et al., EP 0,212,474 entitled BoneMorphogenic Agents.

Wang et al. (Proc. Nat. Acad. Sci. USA (1988) 85: 9484-9488) disclosesthe purification of a bovine bone morphogenetic protein from guanidineextracts of demineralized bone having cartilage and bone formationactivity as a basic protein corresponding to a molecular weight of 30 kDdetermined from gel elution. Purification of the protein yieldedproteins of 30, 18 and 16 kD which, upon separation, were inactive. Inview of this result, the authors acknowledged that the exact identity ofthe active material had not been determined.

Wozney et al. (Science (1988) 242: 1528-1534) discloses the isolation offull-length cDNA's encoding the human equivalents of three polypeptidesoriginally purified from bovine bone. The authors report that each ofthe three recombinantly expressed human proteins are independently or incombination capable of inducing cartilage formation. No evidence of boneformation is reported.

It is an object of this invention to provide osteogenic devicescomprising matrices containing dispersed osteogenic protein capable ofbone induction in allogenic and xenogenic implants. Another object is toprovide a reproducible method of isolating osteogenic protein frommammalian bone tissue. Another object is to characterize the proteinresponsible for osteogenesis. Another object is to provide natural andrecombinant osteogenic proteins capable of inducing endochondral boneformation in mammals, including humans. Yet another object is to providegenes encoding native and non-native osteogenic proteins and methods fortheir production using recombinant DNA techniques. Another object is toprovide novel biosynthetic forms of osteogenic proteins and a structuraldesign for novel, functional osteogenic proteins. Another object is toprovide a suitable deglycosylated collagenous bone matrix as a carrierfor osteogenic protein for use in zenogenic implants. Another object isto provide methods for inducing cartilage formation.

These and other objects and features of the invention will be apparentfrom the description, drawings, and claims which follow.

SUMMARY OF THE INVENTION

This invention involves osteogenic devices which, when implanted in amammalian body, can induce at the locus of the implant the fulldevelopmental cascade of endochondral bone formation and bone marrowdifferentiation. Suitably modified as disclosed herein, the devices alsomay be used to induce cartilage formation. The devices comprise acarrier material, referred to herein as a matrix, having thecharacteristics disclosed below, containing dispersed osteogenic proteineither in its native form or in the form of a biosynthetic construct.

A key to these developments was the elucidation of amino acid sequenceand structure data of native osteogenic protein. A protocol wasdeveloped which results in retrieval of active, substantially pureosteogenic protein from mammalian bone. Investigation of the propertiesand structure of the native form osteogenic protein then permitted theinventors to develop a rational design for non-native forms, i.e., formsnever before known in nature, capable of inducing bone formation. As faras applicants are aware, the constructs disclosed herein constitute thefirst instance of the design of a functional, active protein withoutpreexisting knowledge of the active region of a native form nucleotideor amino acid sequence.

A series of consensus DNA sequences were designed with the goal ofproducing an active osteogenic protein. The sequences were based onpartial amino acid sequence data obtained from the natural sourceproduct and on observed homologies with unrelated genes reported in theliterature, or the sequences they encode, having a presumed ordemonstrated developmental function. Several of the biosyntheticconsensus sequences have been expressed as fusion proteins inprocaryotes, purified, cleaved, refolded, combined with a matrix,implanted in an established animal model, and shown to have endochondralbone-inducing activity. The currently preferred active totallybiosynthetic proteins comprise two synthetic sequences designated COP5and COP7. The amino acid sequences of these proteins are set forthbelow.

1       10         20        30        40 COP5     LYVDFS-DVGWDDWIVAPPGYQAFYCHGECPFPLAD           50        60         70HFNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA     80        90       100ISMLYLDENEKVVLKNYQEMVVEGCGCR (SEQ ID NO: 1)1       10         20        30        40 COP7     LYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD           50        60         70HLNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA     80        90       100ISMLYLDENEKVVLKNYQEMVVEGCGCR (SEQ ID NO: 2)

In these sequences and all other amino acid sequences disclosed herein,the dashes (−) are used as fillers only to line up comparable sequencesin related proteins, and have no other function. Thus, amino acids 45-50of COP7, for example, are NHAVV. Also, the numbering of amino acids isselected solely for purposes of facilitating comparisons betweensequences. Thus, for example, the DF residues numbered at 9 and 10 ofCOP5 and COP7 may comprise residues, e.g., 35 and 36, of an osteogenicprotein embodying invention.

Thus, in one aspect, the invention comprises a protein comprising anamino acid sequence sufficiently duplicative of the sequence of COP5 orCOP7 such that it is capable of inducing endochondral bone formationwhen properly folded and implanted in a mammal in association with amatrix. Some of these sequences induce cartilage, but not bone. Also,the bone forming materials may be used to produce cartilage if implantedin an avascular locus, or if an inhibitor to full bone development isimplanted together with the active protein. Thus, in another aspect, theinvention comprises a protein less than about 200 amino acids long in asequence sufficiently duplicative of the sequence of COP5 or COP7 suchthat it is capable at least of cartilage formation when properly foldedand implanted in a mammal in association with a matrix.

In one preferred aspect, these proteins comprise species of the genericamino acid sequences (SEQ ID NO: 3):

 1       10        20        30        40        50     LXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAXX        60        70         80        90QXXVXXXNXXXXPXXCCXPXXXXXXXXXLXXXXXXXVXLXXYXXMXVX 100 XCXCX or 1       10        20        30        40        50CXXXXLXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAXX        60        70         80        90QXXVXXXNXXXXPXXCCXPXXXXXXXXXLXXXXXXXVXLXXYXXMXVX 100 XCXCX (SEQ ID NO:4)where the letters indicate the amino acid residues of standard singleletter code, and the Xs represent amino acid residues. Preferred aminoacid sequences within the foregoing generic sequences are:

 1       10        20        30        40        50     LYVDFRDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV       K S S L  QE VISE FD Y  E A AY MPESMKAS   VI       F E K I  DN     L    N  S   Q  ITK FP    TL           A    S      K         60        70        80        90QTLVNSVNPGKIPKACCVPTELSAISMLYLDENENVVLKNYQDMVVE  SI HAI SEQVEP  A  EQMNSLAI FFNDQDK I RK EE T     RF    T   S     K DPV V  Y NS     H RN   R      N    S                      K       P 100 GCGCR DA HH S E (SEQ ID NO: 5) and 1       10        20        30        40        50CKRHPLYVDFRDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV  RRRS K S S L  QE VISE FD Y  E A AY MPESMKS    VI    KE F E K I  DN     L    N  S   Q  ITK FP    TL     Q     A    S      K         60        70        80      90QTLVNSVNPGKIPKACCVPTELSAISMLYLDENENVVLKNYQDMVVE  SI HAI SEQVEP  A  EQMNSLAI FFNDQDK I RK EE T     RF    T   S     K DPV V  Y NS     H RN   R      N    S                      K       P 100 GCGCR DA HH S E (SEQ ID NO: 6)wherein each of the amino acids arranged vertically at each position inthe sequence may be used alternatively in various combinations. Notethat these generic sequences have 6 and preferably 7 cysteine residueswhere inter- or intramolecular disulfide bonds can form, and containother critical amino acids which influence the tertiary structure of theproteins. These generic structural features are found in previouslypublished sequences, none of which have been described as capable ofosteogenic activity, and most of which never have been linked with suchactivity.

Particular useful sequences include:

1      10         20        30        40 Vg1CKKRHLYVEFK-DVGWQNWVIAPQGYMANYCYGECPYPLTE          50        60         70 ILNGSN--H-AILQTLVHSIEPED-IPLPCCVPTKMSP    80        90       100 ISMLFYDNNDNVVLRHYENMAVDECGCR (SEQ ID NO: 7)1       10         20        30        40 DPPCRRHSLYVDFS-DVGWDDWIVAPLGYDAYYCHGKCPFPLAD          50         60         70HFNSTN--H-AVVQTLVNNNNPGK-VPKACCVPTQLDS     80        90       100VAMLYLNDQSTVVLKNYQEMTVVGCGCR (SEQ ID NO: 8)                                   −5                                    HQRQA1       10         20        30        40 OP1CKKHELYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS           50        60         70YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA     80        90       100ISVLYFDDSSNVILKKYRNMVVRACGCH (SEQ ID NO: 9)1       10         20        30        40 CBMP-2aCKRHPLYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD           50        60         70HLHSTN--H-AIVQTLVNSVNS-K-IPKACCVPTELSA     80        90       100ISMLYLDENEKVVLKNYQDMVVEGCGCR (SEQ ID NO: 10)1       10         20        30        40 CBMP-2bCRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGDCPFPLAD           50        60         70HLNSTN--H-AIVQTLVNSVNS-S-IPKACCVPTELSA     80        90       100ISMLYLDEYDKVVLKNYQEMVVEGCGCR (SEQ ID NO: 11)1       10         20        30        40 CBMP-3CARRYLKVDFA-DIGWSEWIISPKSFDAYYCSGACQFPMPK           50        60         70SLKPSN--H-ATIQSIVRAVGVVPGIPEPCCVPEKMSS     80        90       100LSILFFDENKNVVLKVYPNMTVESCACR (SEQ ID NO: 12)1       10         20        30        40 COP1     LYVDFQRDVGWDDWIIAPVDFDAYYCSGACQFPSAD           50        60         70HFNSTN--H-AVVQTLVNNMNPGK-VPKPCCVPTELSA     80        90       100ISMLYLDENSTVVLKNYQEMTVVGCGCR (SEQ ID NO: 13)1       10         20        30        40 COP3     LYVDFQRDVGWDDWIVAPPGYQAFYCSGACQFPSAD           50        60         70HFNSTN--H-AVVQTLVNNMNPGK-VPKPCCVPTELSA     80        90       100ISMLYLDENEKVVLKNYQEMVVEGCGCR (SEQ ID NO: 14)1       10         20        30        40 COP4     LYVDFS-DVGWDDWIVAPPGYQAFYCSGACQFPSAD           50        60         70HFNSTN--H-AVVQTLVNNMNPGK-VPKPCCVPTELSA     80        90       100ISMLYLDENEKVVLKNYQEMVVEGCGCR (SEQ ID NO: 15)                             −10                           PKHHSQRARKKNKN1       10         20        30        40 COP16CRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGECPFPLAD           50        60         70HFNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA     80        90       100ISMLYLDENEKVVLKNYQEMVVEGCGCR (SEQ ID NO: 16)Vg1 is a known Xenopus sequence heretofore not associated with boneformation. DPP is an amino acid sequence encoded by a drosophila generesponsible for development of the dorsoventral pattern. OP1 is a regionof a natural sequence encoded by exons of a genomic DNA sequenceretrieved by applicants. The CBMPs are amino acid sequences comprisingsubparts of mammalian proteins encoded by genomic DNAs and cDNAsretrieved by applicants. The COPs are biosynthetic protein sequencesexpressed by novel consensus gene constructs, designed using thecriteria set forth herein, and not yet found in nature.

These proteins are believed to dimerize during refolding. They appearnot to be active when reduced. Various combinations of species of theproteins, i.e., heterodimers, have activity, as do homodimers. As far asapplicants are aware, the COP5 and COP7 constructs constitute the firstinstances of the design of a bioactive protein without preexistingknowledge of the active region of a native form nucleotide or amino acidsequence.

The invention also provides native forms of osteogenic protein,extracted from bone or produced using recombinant DNA techniques. Thesubstantially pure osteogenic protein may include forms having varyingglycosylation patterns, varying N-termini, a family of related proteinshaving regions of amino acid sequence homology, and active truncated ormutated forms of native protein, no matter how derived. The osteogenicprotein in its native form is glycosylated and has an apparent molecularweight of about 30 kD as determined by SDS-PAGE. When reduced, the 30 kDprotein gives rise to two glycosylated polypeptide chains havingapparent molecular weights of about 16 kD and 18 kD. In the reducedstate, the 30 kD protein has no detectable osteogenic activity. Thedeglycosylated protein, which has osteogenic activity, has an apparentmolecular weight of about 27 kD. When reduced, the 27 kD protein givesrise to the two deglycosylated polypeptides have molecular weights ofabout 14 kD to 16 kD.

Analysis of digestion fragments indicate that the native 30 kDosteogenic protein contains the following amino acid sequences (questionmarks indicate undetermined residues):

(1) S-F-D-A-Y-Y-C-S-G-A-C-Q-F-P-M-P-K; (SEQ ID NO: 17) (2)S-L-K-P-S-N-Y-A-T-I-Q-S-I-V; (SEQ ID NO: 18) (3)A-C-C-V-P-T-E-L-S-A-I-S-M-L-Y-L-D-E-N-E-K; (SEQ ID NO: 19) (4)M-S-S-L-S-I-L-F-F-D-E-N-K; (SEQ ID NO: 20) (5) S-Q-E-L-Y-V-D-F-Q-R; (SEQID NO: 21) (6) F-L-H-C-Q-F-S-E-R-N-S; (SEQ ID NO: 22) (7)T-V-G-Q-L-N-E-Q-S-S-E-P-N-I-Y; (SEQ ID NO: 23) (8) l-y-d-p-m-v-v; (SEQID NO: 24) (9) V-G-V-V-P-G-I-P-E-P-C-C-V-P-E; (SEQ ID NO: 25) (10)V-D-F-A-D-I-G; (SEQ ID NO: 26) (11) V-P-K-P-C-C-A-P-T; (SEQ ID NO: 27)(12) I-N-I-A-N-Y-L; (SEQ ID NO: 28) (13) D-N-H-V-L-T-M-F-P-I-A-I-N; (SEQID NO: 29) (14) D-E-Q-T-L-K-K-A-R-R-K-Q-W-I-?-P; (SEQ ID NO: 30) (15)D-I-G-?-S-E-W-I-I-?-P; (SEQ ID NO: 31) (16)S-I-V-R-A-V-G-V-P-G-I-P-E-P-?-?-V; (SEQ ID NO: 32) (17)D-?-I-V-A-P-P-Q-Y-H-A-F-Y; (SEQ ID NO: 33) (18)D-E-N-K-N-V-V-L-K-V-Y-P-N-M-T-V-E; (SEQ ID NO: 34) (19)S-Q-T-L-Q-F-D-E-Q-T-L-K-?-A-R-?-K-Q; (SEQ ID NO: 35) (20)D-E-Q-T-L-K-K-A-R-R-K-Q-W-I-E-P-R-N-?-A-R-R-Y -L; (SEQ ID NO: 36) (21)A-R-R-K-Q-W-I-E-P-R-N-?-A-?-R-Y-?-?-V-D; (SEQ ID NO: 37) and (22)R-?-Q-W-I-E-P-?-N-?-A-?-?-Y-L-K-V-D-?-A-?-? -G. (SEQ ID NO: 38)

The substantially pure (i.e., free of contaminating proteins having noosteoinductive activity) osteogenic proteins and the synthetics areuseful in clinical applications in conjunction with a suitable deliveryor support system (matrix). The matrix is made up of particles or porousmaterials. The pores must be of a dimension to permit progenitor cellmigration and subsequent differentiation and proliferation. The particlesize should be within the range of 70-850 mm, preferably 70-420 mm. Itmay be fabricated by close packing particulate material into a shapespanning the bone defect, or by otherwise structuring as desired amaterial that is biocompatible (non-inflammatory) and, biodegradable invivo to serve as a “temporary scaffold” and substratum for recruitmentof migratory progenitor cells, and as a base for their subsequentanchoring and proliferation. Currently preferred carriers includeparticulate, demineralized, guanidine extracted, species-specific(allogenic) bone, and particulate, deglycosglated, protein extracted,demineralized, zenogenic bone. Optionally, such xenogenic bone powdermatrices also may be treated with proteases such as trypsin. Otheruseful matrix materials comprise collagen, homopolymers and copolymersof glycolic acid and lactic acid, hydroxyapatite, tricalcium phosphateand other calcium phosphates.

The availability of the protein in substantially pure form, andknowledge of its amino acid sequence and other structural features,enable the identification, cloning, and expression of native genes whichencode osteogenic proteins. When properly modified after translation,incorporated in a suitable matrix, and implanted as disclosed herein,these proteins are operative to induce formation of cartilage andendochondral bone.

The consensus DNA sequences are also useful as probes for extractinggenes encoding osteogenic protein from genomic and cDNA libraries. Oneof the consensus sequences has been used to isolate a heretoforeunidentified genomic DNA sequence, portions of which when ligated encodea protein having a region capable of inducing endochondral boneformation. This protein, designated OP1, has an active region having thesequence set forth below.

1       10         20        30        40 OP1     LYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS           50        60         70YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA     80        90       100ISVLYFDDSSNVILKKYRNMVVRACGCH (SEQ ID NO: 39) A longer active sequenceis:                                    −5                                    HQRQA1       10         20        30        40 OP1CKKHELYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS           50        60         70YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA     80        90       100ISVLYFDDSSNVILKKYRNMVVRACGCH (SEQ ID NO: 9)FIG. 1A discloses the genomic DNA sequence of OP1 (SEQ ID NO: 40).

The probes have also retrieved the DNA sequences identified inPCT/087/01537, referenced above, designated therein as BMPII(b) andBMPIII. The inventors herein have discovered that certain subparts ofthese genomic DNAs, and BMPIIa, from the same publication, when properlyassembled, encode proteins (CBMPIIa, CBMPIIb, and CBMPIII) which havetrue osteogenic activity, i.e., induce the full cascade of events whenproperly implanted in a mammal leading to endochondral bone formation.

Thus, in view of this disclosure, skilled genetic engineers can designand synthesize genes or isolate genes from cDNA or genomic librarieswhich encode appropriate amino acid sequences, and then can express themin various types of host cells, including both procaryotes andeucaryotes, to produce large quantities of active proteins in nativeforms, truncated analogs, muteins, fusion proteins, and other constructscapable of inducing bone formation in mammals including humans.

The osteogenic proteins and implantable osteogenic devices enabled anddisclosed herein will permit the physician to obtain optimal predictablebone formation to correct, for example, acquired and congenitalcraniofacial and other skeletal or dental anomalies (Glowacki et al.(1981) Lancet 1: 959-963). The devices may be used to induce localendochondral bone formation in non-union-fractures as demonstrated inanimal tests, and in other clinical applications including periodontalapplications where bone formation is required. The other potentialclinical application is in cartilage repair, for example, in thetreatment of osteoarthritis.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects of this invention, the various featuresthereof, as well as the invention itself, may be more fully understoodfrom the following description, when read together with the accompanyingdrawings, in which:

FIG. 1A represents the nucleotide sequence of the genomic copy ofosteogenic protein “OP1” gene (SEQ ID NO:40). The unknown region between1880 and 1920 actually represents about 1000 nucleotides.

FIG. 1B is a representation of the hybridization of the consensusgene/probe to the osteogenic protein “OP1” gene (SEQ ID NO:42).

FIG. 2 is a collection of plots of protein concentration (as indicatedby optical absorption) vs elution volume illustrating the results ofbovine osteogenic protein (BOP) fractionation during purification onheparin-Sepharose-I; HAP-Ultragel; sieving gel (Sephacryl 300); andheparin-Sepharose-II;

FIG. 3 is a photographic reproduction of a Coomassie blue stained SDSpolyacrylamide gel of the osteogenic protein under non-reducing (A) andreducing (B) conditions;

FIG. 4 is a photographic reproduction of a Con A blot of an SDSpolyacrylamide gel showing the carbohydrate component of oxidized (A)and reduced (B) 30 kD protein;

FIG. 5 is a photographic reproduction of an autoradiogram of an SDSpolyacrylamide gel of ¹²⁵I-labelled glycosylated (A) and deglycosylated(B) osteogenic protein under non-reducing (1) and reducing (2)conditions;

FIG. 6 is a photographic reproduction of an autoradiogram of an SDSpolyacrylamide gel of peptides produced upon the digestion of the 30 kDosteogenic protein with V-8 protease (B), Endo Lys C protease (C),pepsin (D), and trypsin (E). (A) is control;

FIG. 7 is a collection of HPLC chromatograms of tryptic peptidedigestions of 30 kD BOP (A), the 16 kD subunit (B), and the 18 kDsubunit (C);

FIG. 8 is an HPLC chromatogram of an elution profile on reverse phaseC-18 HPLC of the samples recovered from the second heparin-Sepharosechromatography step (see FIG. 2D). Superimposed is the percent boneformation in each fraction;

FIG. 9 is a gel permeation chromatogram of an elution profile on TSK3000/2000 gel of the C-18 purified osteogenic peak fraction.Superimposed is the percent bone formation in each fraction;

FIG. 10 is a collection of graphs of protein concentration (as indicatedby optical absorption) vs. elution volume illustrating the results ofhuman protein fractionation on Heparin-Sepharose I (A), HAP-Ultragel(B), TSK 3000/2000 (C), and heparin-Sepharose II (D). Arrows indicatebuffer changes;

FIG. 11 is a graph showing representative dose response curves forbone-inducing activity in samples from various purification stepsincluding reverse phase HPLC on C-18 (A), Heparin-Sepharose II (B), TSK3000 (C), HAP-ultragel (D), and Heparin-Sepharose I (E);

FIG. 12 is a bar graph of radiomorphometric analyses of feline bonedefect repair after treatment with an osteogenic device (A), carriercontrol (B), and demineralized bone (C);

FIG. 13 is a schematic representation of the DNA sequence (SEQ ID NO:43)and corresponding amino acid sequence (SEQ ID NO:44) of a consensusgene/probe for osteogenic protein (COPO);

FIG. 14 is a graph of osteogenic activity vs. Increasing molecularweight showing peak bone forming activity in the 30 kD region of an SDSpolyacrylamide gel;

FIG. 15 is a photographic representation of a Coomassie blue stained SDSgel showing gel purified subunits of the 30 kD protein;

FIG. 16 is a pair of HPLC chromatograms of Endo Asp N proteinase digestsof the 18 kD subunit (A) and 16 kD subunit (B);

FIG. 17 is a photographic representation of the histological examinationof bone implants in the rat model: carrier alone (A); carrier andglycosylated osteogenic protein (B); and carrier and deglycosylatedosteogenic protein (C). Arrows indicate osteoblasts;

FIG. 18 is a comparison of the amino acid sequence of various osteogenicproteins to those of the TGF-beta family. COP1 (SEQ ID NO: 13), COP3(SEQ ID NO: 14), COP4 (SEQ ID NO: 15), COP5 (SEQ ID NO: 1), and COP7(SEQ ID NO: 2) are a family of analogs of synthetic osteogenic proteinsdeveloped from the consensus gene that was joined to a leader proteinvia a hinge region having the sequence D-P-N-G (SEQ ID NO: 45) thatpermitted chemical cleavage at the D-P site (by acid) or N-G (byhydroxylamine) resulting in the release of the analog protein; VGI (SEQID NO: 7) is a Xenopus protein, DPP (SEQ ID NO: 8) is a Drosophilaprotein; OP1 (amino acids 6-x of SEQ ID NO: 9) is a native osteogenicprotein; CBMP2a (SEQ ID NO: 10) and 2b (SEQ ID NO: 11), and CBMP3 (SEQID NO: 12) are subparts of proteins disclosed in PCT application087/01537; beta-Inhibin a) is shown in SEQ ID NO: 46, beta-Inhibin b) isshown in SEQ ID NO: 47, TGF-beta 1 is shown in SEQ ID NO: 48, TGF-beta 2is shown in SEQ ID NO: 49, TGF-beta 3 is shown in SEQ ID NO: 50; MIS(SEQ ID NO: 51) is Mullerian inhibitory substance; alpha-Inhibin isshown in SEQ ID NO: 52; and “consensus choices” represent varioussubstitutions of amino acids that may be made at various positions inosteogenic proteins;

FIG. 19 is a graph illustrating the activity of xenogenic matrix(deglycosylated bovine matrix);

FIGS. 20A and 20B are bar graphs showing the specific activity ofnaturally sourced OP before and after gel elution as measured by calciumcontent vs. increasing concentrations of proteins (dose curve, in ng);

FIG. 21A is an E. coli expression vector containing a gene of anosteogenic protein fused to a leader protein;

FIG. 21B is the DNA sequence (SEQ ID NO:53) and amino acid sequence (SEQID NO:54) comprising a modified trp-LE leader, two Fb domains of proteinA, an ASP-PRO cleavage site, and the COP5 sequence.

FIGS. 22A and 22B are photomicrographs of implants showing the histology(day 12) of COP5 active recombinant protein. A is a control (rat matrixalone, 25 mg). B is rat matrix plus COP5, showing +++ cartilageformation and ++ bone formation (see key infra). Similar results areachieved with COP7.

DESCRIPTION

Purification protocols have been developed which enable isolation of theosteogenic protein present in crude protein extracts from mammalianbone. While each of the separation steps constitute known separationtechniques, it has been discovered that the combination of a sequence ofseparations exploiting the protein's affinity for heparin and forhydroxyapatite (HAP) in the presence of a denaturant such as urea is keyto isolating the pure protein from the crude extract. These criticalseparation steps are combined with separations on hydrophobic media, gelexclusion chromatography, and elution form SDS PAGE.

The isolation procedure enables the production of significant quantitiesof substantially pure osteogenic protein from any mammalian species,provided sufficient amounts of fresh bone from the species is available.The empirical development of the procedure, coupled with theavailability of fresh calf bone, has enabled isolation of substantiallypure bovine osteogenic protein (BOP). BOP has been characterizedsignificantly as set forth below; its ability to induce cartilage andultimately endochondral bone growth in cat, rabbit, and rat have beenstudied; it has been shown to be able to induce the full developmentalcascade of bone formation previously ascribed to unknown protein orproteins in heterogeneous bone extracts; and it may be used to induceformation of endochondral bone in orthopedic defects including non-unionfractures. In its native form it is a glycosylated, dimeric protein.However, it is active in deglycosylated form. It has been partiallysequenced. Its primary structure includes the amino acid sequences setforth herein.

Elucidation of the amino acid sequence of BOP enables the constructionof pools of nucleic acid probes encoding peptide fragments. Also, aconsensus nucleic acid sequence designed as disclosed herein based onthe amino acid sequence data, inferred codons for the sequences, andobservation of partial homology with known genes, also may be used as aprobe. The probes may be used to isolate naturally occurring cDNAs whichencode active mammalian osteogenic proteins (OP) as described belowusing standard hybridization methodology. The mRNAs are present in thecytoplasm of cells of various species which are known to synthesizeosteogenic proteins. Useful cells harboring the mRNAs include, forexample, osteoblasts from bone or osteosarcoma, hypertrophicchondrocytes, and stem cells. The mRNAs can be used to produce cDNAlibraries. Alternatively, relevant DNAs encoding osteogenic protein maybe retrieved from cloned genomic DNA libraries from various mammalianspecies.

The consensus sequence described above also may be refined by comparisonwith the sequences present in certain regulatory genes from drosophila,zenopus, and human followed by point mutation, expression, and assay foractivity. This approach has been successful in producing several activetotally synthetic constructs not found in nature (as far as applicantsare aware) which have true osteogenic activity.

These discoveries enable the construction of DNAS encoding totallynovel, non-native protein constructs which individually, and combinedare capable of producing true endochondral bone. They also permitexpression of the natural material, truncated forms, muteins, analogs,fusion proteins, and various other variants and constructs, from cDNAsretrieved from natural sources or synthesized using the techniquesdisclosed herein using automated, commercially available equipment. TheDNAs may be expressed using well established recombinant DNAtechnologies in procaryotic or eucaryotic host cells, and may beoxidized and refolded in vitro if necessary for biological activity.

The isolation procedure for obtaining the protein from bone, theretrieval of an osteogenic protein gene, the design and production ofbiosynthetics, the nature of the matrix, and other material aspectsconcerning the nature, utility, how to make, and how to use the subjectmatter claimed herein will be further understood from the following,which constitutes the best method currently known for practicing thevarious aspects of the invention.

I. Naturally Sourced Osteogenic Protein

A—PURIFICATION

A1. Preparation of Demineralized Bone

Demineralized bovine bone matrix is prepared by previously publishedprocedures (Sampath and Reddi (1983) Proc. Natl. Acad. Sci. USA 80:6591-6595). Bovine diaphyseal bones (age 1-10 days) are obtained from alocal slaughterhouse and used fresh. The bones are stripped of muscleand fat, cleaned of periosteum, demarrowed by pressure with cold water,dipped in cold absolute ethanol, and stored at −20° C. They are thendried and fragmented by crushing and pulverized in a large mill. Care istaken to prevent heating by using liquid nitrogen. The pulverized boneis milled to a particle size between 70-420 mm and is defatted by twowashes of approximately two hours duration with three volumes ofchloroform and methanol (3:1). The particulate bone is then washed withone volume of absolute ethanol and dried over one volume of anhydrousether. The defatted bone powder (the alternative method is to obtainBovine Cortical Bone Powder (75-425 mm) from American Biomaterials) isthen demineralized with 10 volumes of 0.5 N HCl at 4° C. for 40 min.,four times. Finally, neutralizing washes are done on the demineralizedbone powder with a large volume of water.

A2. Dissociative Extraction and Ethanol Precipitation

Demineralized bone matrix thus prepared is dissociatively extracted with5 volumes of 4 M guanidine-HCl, 50M Tris-HCl, pH 7.0, containingprotease inhibitors (5 mM benzamidine, 44 mM 6-aminohexanoic acid, 4.3mM N-ethylmaleimide, 0.44 mM phenylmethylsulfonyfluoride) for 16 hr. at4° C. The suspension is filtered. The supernatant is collected andconcentrated to one volume using an ultrafiltration hollow fibermembrane (Amicon, YM-10). The concentrate is centrifuged (8,000×g for 10min. at 4° C.), and the supernatant is then subjected to ethanolprecipitation. To one volume of concentrate is added five volumes ofcold (−70° C.) absolute ethanol (100%), which is then kept at −70° C.for 16 hrs. The precipitate is obtained upon centrifugation at 10,000×9for 10 min. at 4° C. The resulting pellet is resuspended in 4 l of 85%cold ethanol incubated for 60 min. at −70° C. and recentrifuged. Theprecipitate is again resuspended in 85% cold ethanol (2 l), incubated at−70° C. for 60 min. and centrifuged. The precipitate is thenlyophilized.

A3. Heparin-Sepharose Chromatography I

The ethanol precipitated, lyophilized, extracted crude protein isdissolved in 25 volumes of 6 M urea, 50 mM Tris-HCl, pH 7.0 (Buffer A)containing 0.15 M lacI, and clarified by centrifugation at 8,000×g for10 min. The heparin-Sepharose is column-equilibrated with Buffer A. Theprotein is loaded onto the column and after washing with three columnvolume of initial buffer (Buffer A containing 0.15 M HaCl), protein iseluted with Buffer A containing 0.5 M NaCl. The absorption of the eluateis monitored continuously at 280 nm. The pool of protein eluted by 0.5 MNaCl (approximately 1 column volumes) is collected and stored at 4° C.

As shown in FIG. 2A, most of the protein (about 95%) remains unbound.Approximately 5% of the protein is bound to the column. The unboundfraction has no bone inductive activity when bioassayed as a whole orafter a partial purification through Sepharose CL-6B.

A4. Hydroxyapaptite-Ultrogel Chromatography

The volume of protein eluted by Buffer A containing 0.5 M NaCl from theheparin-Sepharose is applied directly to a column ofhydroxyapaptite-ultrogel (HAP-ultrogel) (LKB Instruments), equilibratedwith Buffer A containing 0.5 M NaCl. The HAP-ultrogel is treated withBuffer A containing 500 mM Na phosphate prior to equilibration. Theunadsorbed protein is collected as an unbound fraction, and the columnis washed with three column volumes of Buffer A containing 0.5H NaCl.The column is subsequently eluted with Buffer A containing 100 mM NaPhosphate (FIG. 2B).

The eluted component can induce endochondral bone as measured byalkaline phosphatase activity and histology. As the biologically activeprotein is bound to HAP in the presence of 6 M urea and 0.5 MN NaCl, itis likely that the protein has an affinity for bone mineral and may bedisplaced only by phosphate ions.

A5. Sephacryl S-300 Gel Exclusion Chromatography

Sephacryl S-300 HR (High Resolution, 5 cm×100 cm column) is obtainedfrom Pharmacia and equilibrated with 4 M guanidine-HCl, 50 mM Tris-HCl,pH 7.0. The bound protein fraction from HA-ultrogel is concentrated andexchanged from urea to 4 M guanidine-HCl, 50M Tris-HCl, pH 7.0 via anAmicon ultrafiltration YM-10 membrane. The solution is then filteredwith Schleicher and Schuell CENTREX disposable microfilters. A samplealiquot of approximately 15 ml containing approximately 400 mg ofprotein is loaded onto the column and then eluted with 4 Mguanidine-HCl, 50 mM Tris-HCl, pH 7.0, with a flow rate of 3 ml/min; 12ml fractions are collected over 8 hours and the concentration of proteinis measured at A₂₈₀ nm (FIG. 2C). An aliquot of the individual fractionsis bioassayed for bone formation. Those fractions which have shown boneformation and have a molecular weigh less than 35 kD are pooled andconcentrated via an Amicon ultrafiltration system with YM-10 membrane.

A6. Heparin-Sepharose Chromatography-II

The pooled osteo-inductive fractions obtained from gel exclusionchromatography are dialysed extensively against distilled water and thenagainst 6 M urea, 50 mM Tris-HCl, pH 7.0 (Buffer A) containing 0.1 MNaCl. The dialysate is then cleared through centrifugation. The sampleis applied to the heparin-sepharose column (equilibrated with the samebuffer). After washing with three column volumes of initial buffer, thecolumn is developed sequentially with Buffer B containing 0.15 M MaCl,and 0.5 N NaCl (FIG. 2D). The protein eluted by 0.5 M NaCl is collectedand dialyzed extensively against distilled water. It is then dialyzedagainst 30% acetonitrile, 0.1% TFA at 4° C.

A7. Reverse Phase HPLC

The protein is further purified by C-18 Vydac silica-based HPLC columnchromatography (particle size 5 mm; pore size 300 A). The osteoinductivefraction obtained from heparin-sepharose-II chromatograph is loaded ontothe column, and washed in 0.1% TFA, 10% acetonitrile for five min. Asshown in FIG. 8, the bound proteins are eluted with a linear gradient of10-30% acetonitrile over 15 min., 30-50% acetonitrile over 60 min, and50-70% acetonitrile over 10 min at 22° C. with a flow rate of 1.5 ml/minand 1.4 ml samples are collected in polycarbonate tubes. Protein ismonitored by absorbance at A₂₁₄ nm. Column fractions are tested for thepresence of osteoinductive activity, concanavalin A-blottable proteinsand then pooled. Pools are then characterized biochemically for thepresence of 30 kD protein by autoradiography, concanavalin A blotting,and Coomassie blue dye staining. They are then assayed for in vivoosteogenic activity. Biological activity is not found in the absence of30 kD protein.

A8. Gel Elution

The glycosylated or deglycosylated protein is eluted from SDS gels (0.5mm and 1.5 mm thickness) for further characterization. ¹²⁵I-labelled 30kD protein is routinely added to each preparation to monitor yields.TABLE 1 shows the various elution buffers that have been tested and theyields of ¹²⁵I-labelled protein.

TABLE 1 Elution of 30 kD Protein from SDS Gel % Eluted Buffer 0.5 mm 1.5mm (1) dH₂O 22 (2) 4M Guanidine-HCl, Tris-HCl, pH 7.0 2 (3) 4MGuanidine-HCl, Tris-HCl, pH 7.0, 93 52 0.5% Triton × 100 (4) 0.1% SDS,Tris-HCl, pH 7.0 98

TABLE 2 lists the steps used to isolate the 30 kD or deglycosylated 27kD gel-bound protein. The standard protocol uses diffusion elution using4M guanidine-HCl containing 0.5% Triton x 100 in Tris-HCl buffer or inTris-HCl buffer containing 0.1% SDS to achieve greater than 95% elutionof the protein from the 27 or 30 kD region of the gel for demonstrationof osteogenic activity in vivo as described in later section.

In order to isolate substantially purified 30 kD or deglycosylated 27 kDprotein for sequencing and characterization, the following steps arementioned in Table 2.

TABLE 2 Preparation of Gel Eluted Protein (C-18 Pool or deglycoslatedprotein plus ¹²⁵I-labelled 30 kD protein) 1. Dry using vacuumcentrifugation; 2. Wash pellet with H₂0; 3. Dissolve pellet in gelsample buffer (no reducing agent); 4. Electrophorese onpre-electrophoresed 0.5 mm mini gel; 5. Cut out 27 or 30 kD protein; 6.Elute from gel with 0.1% SDS, 50 mM Tris-HCl, pH 7.0; 7. Filter throughCentrex membrane; 8. Concentrate in Centricon tube (10 kD membrane); 9.Chromatograph of TSK-3000 gel filtration column; 10. Concentrate inCentricon tube.

Chromatography in 0.1% SDS on a TSK-3000 gel filtration column isperformed to separate gel impurities, such as soluble acrylamide, fromthe final product. The overall yield of labelled 30 kD protein from thegel elution protocol is 50-60% of the loaded sample. Most of the lossoccurs in the electrophoresis step, due to protein aggregation and/orsmearing. In a separate experiment, a sample of gel eluted 30 kD proteinis reduced, electrophoresed on an SDS gel, and transferred to anImmobilon membrane. The membrane is stained with Coomassie blue dye, cutinto slices, and the slices are counted. Coomassie blue dye stains the16 kD and 18 kD reduced species of the 30 kD protein almost exclusively.However, the counts showed significant smearing throughout the gel inaddition to being concentrated in the 16 kD and 18 kD species. Thissuggests that the ¹²⁵I-label can exhibit anomolous behavior on SDS gelsand cannot be used as an accurate marker for cold protein under suchcircumstances.

The yield is 0.5 to 1.0 mg substantially pure osteogenic protein per kgof bone.

A9. Isolation of the 16 kD and 18 kD Species

TABLE 3 summarizes the procedures involved in the preparation of thesubunits. Approximately 10 mg of gel eluted 30 kD protein (FIG. 3) iscarboxymethylated and electrophoresed on an SDS-gel.

The sample contains ¹²⁵I-label to trace yields and to use as anindicator for slicing the 16 kD, IS kD and non-reduceable 30 K regionsfrom the gel. FIG. 15 shows a Coomassie stained gel of aliquots of theprotein isolated from the different gel slices. The slices correspondingto the 16 kD, 18 kD and non-reduceable 30 kD species containedapproximately 2-3 mg, 3-4 mg, and 1-2 mg, of protein respectively, asestimated by staining intensity.

Prior to SDS electrophoresis, all of the 30 kD species can be reduced tothe 16 kD and 18 kID species. The nonreducible 30 kD species observedafter electrophoresis appears to be an artifact resulting from theelectrophoresis procedure.

TABLE 3 Isolation of the Subunits of the 30 kD protein (C-18 pool plus¹²⁵I labeled 30 kD protein) 1. Electrophorese on SDS gel. 2. Cut out 30kD protein. 3. Elute with 0.1% SDS, 50 nm Tris, pH 7.0. 4. Concentrateand wash with H₂O in Centricon tube (10 kD membranes). 5. Reduce andcarboxymethylate in 1% SDS, 0.4 M Tris, pH 8.5. 6. Concentrate and washwith H₂O in Centricon tube. 7. Electrophorese on SDS gel. 8. Cut out the16 kD and 18 kD subunits. 9. Elute with 0.1% SDS, 50 mM Tris, pH 7.0.10. Concentrate and wash with H₂O in Centricon tubes.B. Demonstration that the 30 KD Protein is Osteogenic Protein—BiologicalCharacterizationB1. Gel Slicing:

Gel slicing experiments confirm that the isolated 30 kD protein is theprotein responsible for osteogenic activity.

Gels from the last step of the purification are sliced. Protein in eachfraction is extracted in 15 mM Tris-HCl, pH 7.0 containing 0.1% SDS orin buffer containing 4M guanidine-HCl, 0.5% non-ionic detergent (Tritonx 100), 50 mM Tris-HCl. The extracted proteins are desalted,concentrated, and assayed for endochondral bone formation activity. Theresults are set forth in FIG. 14. From this Figure it is clear that themajority of osteogenic activity is due to protein at 30 kD region of thegene. Activity in higher molecular weight regions is apparently due toprotein aggregation. These protein aggregates, when reduced, yields the16 kD and 18 kD species discussed above.

B2. Con A-Sepharose Chromatography:

A sample containing the 30 kD protein is solubilized using 0.1% SDS, 50mM Tris-HCl, and is applied to a column of Con A-Sepharose equilibratedwith the same buffer. The bound material is eluted in SDS Tris-HClbuffer containing 0.5 M alpha-methyl mannoside. After reverse phasechromatography of both the bound and unbound fractions, Con A-boundmaterials, when implanted, result in extensive bone formation. Furthercharacterization of the bound materials show a Con A-blottable 30 kDprotein. Accordingly, the 30 kD glycosylated protein is responsible forthe bone forming activity.

B3. Gel Permeation Chromatography:

TSK-3000/2000 gel permeation chromatography in guanidine-HCl alternatelyis used to achieve separation of the high specific activity fractionobtained from C-18 chromatography (FIG. 9). The results demonstrate thatthe peak of bone inducing activity elutes in fractions containingsubstantially pure 30 kD protein by Coomassie blue staining. When thisfraction is iodinated and subjected to autoradiography, a strong band at30 kD accounts for 90% of the iodinated proteins. The fraction inducesbone formation in vivo at a dose of 50 to 100 ng per implant.

B4. Structural Requirements for Biological Activity

Although the role of 30 kD osteogenic protein is clearly established forbone induction, through analysis of proteolytic cleavage products wehave begun to search for a minimum structure that is necessary foractivity in vivo. The results of cleavage experiments demonstrate thatpepsin treatment fails to destroy bone inducing capacity, whereastrypsin or CNBr completely abolishes the activity.

An experiment is performed to isolate and identify pepsin digestedproduct responsible for biological activity. Sample used for pepsindigest were 20%-30% pure. The buffer used is 0.1 TFA in water. Theenzyme to substrate ratio is 1:10. A control sample is made withoutenzyme. The digestion mixture is incubated at room temperature for 16hr. The digested product is then separated in 4 M guanidine-HCl usinggel permeation chromatography, and the fractions are prepared for invivo assay. The results demonstrate that active fractions from gelpermeation chromatography of the pepsin digest correspond to molecularweight of 8 kD-10 kD.

In order to understand the importance of the carbohydrates moiety withrespect to osteogenic activity, the 30 kD protein has been chemicallydeglycosylated using HF (see below). After analyzing an aliquot of thereaction product by Con A blot to confirm the absence of carbohydrate,the material is assayed for its activity in vivo. The bioassay ispositive (i.e., the deglycosylated protein produces a bone formationresponse as determined by histological examination shown in FIG. 17C),demonstrating that exposure to HF did not destroy the biologicalfunction of the protein. In addition, the specific activity of thedeglycosylated protein is approximately the same as that of the nativeglycosylated protein.

B5. Specific Activity of BOP

Experiments were performed 1) to determine the half maximalbone-inducing activity based on calcium content of the implant; 2) toestimate proteins at nanogram levels using a gel scanning method; and 3)to establish dose for half maximal bone inducing activity for gel eluted30 kD BOP. The results demonstrate that gel eluted substantially pure 30kD osteogenic protein induces bone at less than 5 ng per 25 mg implantand exhibits half maximal bone differentiation activity at 20 ng perimplant. The purification data suggest that osteogenic protein has beenpurified from bovine bone to 367,307 fold after final gel elution stepwith a specific activity of 47,750 bone forming units per mg of protein.

B5(a)Half Maximal Bone Differentiation Activity

The bone inducing activity is determined biochemically by the specificactivity of alkaline phosphatase and calcium content of the day 12implant. An increase in the specific activity of alkaline phosphataseindicates the onset of bone formation. Calcium content, on the otherhand, is proportional to the amount of bone formed in the implant. Thebone formation is therefore calculated by determining calcium content ofthe implant on day 12 in rats and expressed as bone forming units, whichrepresent the amount that exhibits half maximal bone inducing activitycompared to rat demineralized bone matrix. Bone induction exhibited byintact demineralized rat bone matrix is considered to be the maximalbone-differentiation activity for comparison.

B5(b)Protein Estimation Using Gel Scanning Techniques

A standard curve is developed employing known amounts of a standardprotein, bovine serum albumin. The protein at varying concentration(50-300 ng) is loaded on 15% SDS gel, electrophoresed, stained incomassie and destained. The gel containing standard proteins is scannedat predetermined settings using a gel scanner at 580 nm. The areacovered by the protein band is calculated and a standard curve againstconcentrations of protein is constructed. A sample with an unknownprotein concentration is electrophoresed with known concentration ofBSA. The lane contained unknown sample is scanned and from the area theconcentration of protein is determined.

B5(c)Gel Elution and Specific Activity

An aliquot of C-18 highly purified active fraction is subjected to SDSgel and sliced according to molecular weights described in FIG. 14.Proteins are eluted from the slices in 4 M guanidine-HCl containing 0.5%Triton X-100, desalted, concentrated and assayed for endochondral boneforming activity as determined by calcium content. The C-18 highlyactive fractions and gel eluted substantially pure 30 kD osteogenicprotein are implanted in varying concentrations in order to determinethe half maximal bone inducing activity.

FIG. 14 demonstrates that the bone inducing activity is due to proteinseluted at 28-34 kD region. The recovery of activity after gel elutionstep is determined by calcium content. FIGS. 20A and 20B represent thebone inducing activity for the various concentrations of 30 kD proteinbefore and after gel elution as estimated by calcium content. Theconcentration of protein is determined by gel scanning in the 30 kDregion. The data suggest that the half maximal activity for 30 kDprotein before gel elution is 69 nanogram per 25 mg implant and is 21nanogram per 25 mg implant after elution. Table 4 describes the yield,total specific activity, and fold purification of osteogenic protein ateach step during purification. Approximately 500 ug of heparin sepharoseI fraction, 130-150 ug of the HA ultrogel fraction, 10-12 ug of the gelfiltration fraction, 4-5 ug of the heparin sepharose II fraction,0.4-0.5 ug of the C-18 highly purified fraction, and 20-25 ng of geleluted substantially purified is needed per 25 mg of implant forunequivocal bone formation for half maximal activity. Thus, 0.8-1.0 ngpurified osteogenic protein per mg. of implant is required to exhibithalf maximal bone differentiation activity in vivo.

TABLE 4 PURIFICATION OF BOP Biological Specific Purification ProteinActivity Activity Purification Steps (mg.) Units* Units/mg. Fold Ethanol30,000# 4,000 0.13 1 Precipitate** Heparin  1,200# 2,400 2.00 15Sepharose I HA-Ultrogel   300# 2,307 7.69 59 Gel filtration    20# 1,60080.00 615 Heparin    5# 1,000 200.00 1,538 Sepharose II C-18 HPLC   0.070@ 150 2,043.00 15,715 Gel elution    0.004@ 191 47,750.00367,307 Values are calculated from 4 kg. of bovine bone matrix (800 g ofdemineralized matrix). *One unit of bone forming activity is defined asthe amount that exhibits half maximal bone differentiation activitycompared to rat demineralized bone matrix, as determined by calciumcontent of the implant on day 12 in rats. #Proteins were measured byabsorbance at 280 nm. @Proteins were measured by gel scanning methodcompared to known standard protein, bovine serum albumin.**Ethanol-precipitated guanidine extract of bovine bone is a weakinducer of bone in rats, possibly due to endogenous inhibitors. Thisprecipitate is subjected to gel filtration and proteins less than 50 kDwere separated and used for bioassay.C. Chemical Characterization of BOPC1. Molecular Weight and structure

Electrophoresis of the most active fractions from reverse phase C-18chromatography on non-reducing SDS polyacrylamide gels reveals a singleband at about 30 kD as detected by both Coomassie blue staining (FIG.3A) and autoradiography.

In order to extend the analysis of BOP, the protein was examined underreducing conditions. FIG. 3B shows an SDS gel of BOP in the presence ofdithiothreitol. Upon reduction, 30 kD BOP yields two species which arestained with Coomassic blue dye: a 16 kD species and an 18 kD species.Reduction causes loss of biological activity. Methods fot the efficientelution of the proteins from SDS gels have been tested, and a protocolhas been developed to achieve purification of both proteins. The tworeduced BOP species have been analyzed to determine if they arestructurally related. Comparison of the amino acid composition of thetwo proteins (as disclosed below) shows little differences, indicatingthat the native protein may comprise two chains having some homology.

C2. Charge Determination

Isoelectric focusing studies are initiated to further evaluate the 30 kDprotein for possible heterogeneity. Results to date have not revealedany such heterogeneity. The oxidized and reduced species migrate asdiffuse bands in the basic region of the isoelectric focusing gel, usingthe iodinated 30 kD protein for detection. Using two dimensional gelelectrophoresis and Con A for detection, the oxidized 30 kD protein showone species migrating in the same basic region as the iodinated 30 kDprotein. The diffuse character of the band may be traced to the presenceof carbohydrate attached to the protein.

C3. Presence of Carbohydrate

The 30 kD protein has been tested for the presence of carbohydrate byConcanavalin A (Con A) blotting after SDS-PAGE and transfer tonitrocellulose paper. The results demonstrate that the 30 kD protein hasa high affinity for Con A, indicating that the protein is glycosylated(FIG. 4A). In addition, the Con A blots provide evidence for asubstructure in the 30 kD region of the gel, suggesting heterogeneitydue to varying degrees of glycosylation. After reduction (FIG. 4B), ConA blots show evidence for two major components at 16 kD and 18 kD. Inaddition, it has been demonstrated that no glycosylated material remainsat the 30 kD region after reduction.

In order to confirm the presence of carbohydrate and to estimate theamount of carbohydrate attached, the 30 kD protein is treated withN-glycanase, a deglycosylating enzyme with a broad specificity. Samplesof the ¹²⁵I-labelled 30 kD protein are incubated with the enzyme in thepresence of SDS for 24 hours at 37° C. As observed by SDS-PAGE, thetreated samples appear as a prominent species at about 27 kD (FIG. 5A).Upon reduction, the 27 kD species is reduced to species having amolecular weight of about 14 kD-16 kD (FIG. 53).

Chemical cleavage of the carbohydrate moieties using hydrogen fluoride(HP) is performed to assess the role of carbohydrate on the boneinducing activity of BOP in vivo. Active osteogenic protein fractionspooled from the C-18 chromatography step are dried in vacuo over P₂O₅ ina polypropylene tube, and 50 ml freshly distilled anhydrous HF at −70°C. is added. After capping the tube tightly, the mixture is kept at 0°C. in an ice-bath with occasional agitation for 1 hr. The HF is thenevaporated using a continuous stream of dry nitrogen gas. The tube isremoved from the ice bath and the residue dried in vacuo over P₂O₅ andKOH pellets.

Following drying, the samples are dissolved in 100 ml of 50%acetonitrile/0.1% TFA and aliquoted for SDS gel analysis, Con A binding,and biological assay. Aliquots are dried and dissolved in either SDS gelsample buffer in preparation for SDS gel analysis and Con A blotting or4 M guanidine-HCl, 50 mM Tris-HCl, pH 7.0 for biological assay.

The results show that samples are completely deglycosylated by the HFtreatment: Con A blots after SDS gel electrophoreses and transfer toImmobilon membrane showed no binding of Con A to the treated samples,while untreated controls were strongly positive at 30 kD. Coomassie gelsof treated samples showed the presense of a 27 kD band instead of the 30kD band present in the untreated controls.

C4. Chemical and Enzymatic Cleavage

Cleavage reactions with CNBr are analyzed using Con A binding fordetection of fragments associated with carbohydrate. Cleavage reactionsare conducted using trifluoroacetic acid (TFA) in the presence andabsence of CNBr. Reactions are conducted at 37° C. for 18 hours, and thesamples are vacuum dried. The samples are washed with water, dissolvedin SDS gel sample buffer with reducing agent, boiled and applied to anSDS gel. After electrophoresis, the protein is transferred to Immobilonmembrane and visualized by Con A binding. In low concentrations of acid(1%), CNBr cleaves the majority of 16 kD and 18 kD species to oneproduct, a species about 14 kD. In reactions using 10% TFA, a 14 kDspecies is observed both with and without CNBr.

Four proteolytic enzymes are used in these experiments to examine thedigestion products of the kD protein: 1) V-8 protease; 2) Endo Lys Cprotease; 3) pepsin; and 4) trypsin. Except for pepsin, the digestionbuffer for the enzymes is 0.1 M ammonium bicarbonate, pH 8.3. The pepsinreactions are done in 0.1% TFA. The digestion volume is 100 ml and theratio of enzyme to substrate is 1:10. ¹²⁵I-labelled 30 kD osteogenicprotein is added for detection. After incubation at 37° C. for 16 hr.,digestion mixtures are dried down and taken up in gel sample buffercontaining dithiothreitol for SDS-PAGE. FIG. 6 shows an autoradiographof an SDS gel of the digestion products. The results show that underthese conditions, only trypsin digests the reduced 16 kD/18 kD speciescompletely and yields a major species at around 12 kD. Pepsin digestionyields better defined, lower molecular weight species. However, the 16kD/18 kD fragments were not digested completely. The V-8 digest showslimited digestion with one dominant species at 16 kD.

C5. Protein Sequencing

To obtain amino acid sequence data, the protein is cleaved with trypsinor Endoproteinase Asp-N (EndoAsp-N). The tryptic digest of reduced andcarboxymethylated 30 kD protein (approximately 10 mg) is fractionated byreverse-phase HPLC using a C-8 narrowbore column (13 cm×2.1 mm ID) witha TFA/acetonitrile gradient and a flow rate of 150 ml/min. The gradientemploys (A) 0.06% TFA in water and (B) 0.04% TFA in water andacetonitrile (1:4; v:v). The procedure was 10% B for five min., followedby a linear gradient for 70 min. to 80% B, followed by a linear gradientfor 10 min. to 100% B. Fractions containing fragments as determined fromthe peaks in the HPLC profile (FIG. 7A) are rechromatographed at leastonce under the same conditions in order to isolate single componentssatisfactory for sequence analysis.

The HPLC profiles of the similarly digested 16 kD and 18 kD subunits areshown in FIGS. 7B and 7C, respectively. These peptide maps are similarsuggesting that the subunits are identical or are closely related.

The 16 kD and 18 kD subunits are digested with Endo Asp N proteinase.The protein is treated with 0.5 mg EndoAsp-N in 50 mM sodium phosphatebuffer, pH 7.8 at 36° C. for 20 hr. The conditions for fractionation arethe same as those described previously for the 30 kD, 16 kD, and 18 kDdigests. The profiles obtained are shown in FIGS. 16A and 16B.

Various of the peptide fragments produced using the foregoing procedureshave been analyzed in an automated amino acid sequencer (AppliedBiosystems 470A with 120A on-line PTH analysis). The following sequencedata has been obtained:

(1) S-F-D-A-Y-Y-C-S-G-A-C-Q-F-P-M-P-K; (SEQ ID NO: 17) (2)S-L-K-P-S-N-Y-A-T-I-Q-S-I-V; (SEQ ID NO: 18) (3)A-C-C-V-P-T-E-L-S-A-I-S-M-L-Y-L-D-E-N-E-K; (SEQ ID NO: 19) (4)M-S-S-L-S-I-L-F-F-D-E-N-K; (SEQ ID NO: 20) (5) S-Q-E-L-Y-V-D-F-Q-R; (SEQID NO: 21) (6) F-L-H-C-Q-F-S-E-R-N-S; (SEQ ID NO: 22) (7)T-V-G-Q-L-N-E-Q-S-S-E-P-N-I-Y; (SEQ ID NO: 23) (8) L-Y-D-P-M-V-V; (SEQID NO: 24) (9) V-G-V-V-P-G-I-P-E-P-C-C-V-P-E; (SEQ ID NO: 25) (10)V-D-F-A-D-I-G; (SEQ ID NO: 26) (11) V-P-K-P-C-C-A-P-T; (SEQ ID NO: 27)(12) I-N-I-A-N-Y-L; (SEQ ID NO: 28) (13) D-N-H-V-L-T-M-F-P-I-A-I-N; (SEQID NO: 29) (14) D-E-Q-T-L-K-K-A-R-R-K-Q-W-I-?-P; (SEQ ID NO: 30) (15)D-I-G-?-S-E-W-I-I-?-P; (SEQ ID NO: 31) (16)S-I-V-R-A-V-G-V-P-G-I-P-E-P-?-?-V; (SEQ ID NO: 32) (17)D-?-I-V-A-P-P-Q-Y-H-A-F-Y; (SEQ ID NO: 33) (18)D-E-N-K-N-V-V-L-K-V-Y-P-N-M-T-V-E; (SEQ ID NO: 34) (19)S-Q-T-L-Q-F-D_E-Q-T-L-K-?-A-R-?-K-Q; (SEQ ID NO: 35) (20)D-E-Q-T-L-K-K-A-R-R-K-Q-W-I-E-P-R-N-?-A-R-R-Y -L; (SEQ ID NO: 36) (21)A-R-R-K-Q-W-I-E-P-R-N-?-A-?-R-Y-?-?-V-D; (SEQ ID NO: 37) and (22)R-?-Q-W-I-E-P-?-N-?-A-?-?-Y-L-K-V-D-?-A-?-? -G. (SEQ ID NO: 38)

Strategies for obtaining amino acid composition were developed using gelelution from 15% SDS gels, transfer onto Immobilon, and hydrolysisImmobilon membrane is a polymer of vinylidene difluoride and, therefore,is not susceptible to acid cleavage. Samples of oxidized (30 kD) andreduced (16 kD and 18 kD) BOP are electrophoresed on a gel andtransferred to Immobilon for hydrolysis and analysis as described below.The composition date generated by amino acid analyses of 30 kD BOP isreproducible, with some variation in the number of residues for a fewamino acids, especially cysteine and isoleucine.

Samples are run on 15% SDS gels, transferred to Immobilon, and stainedwith Coomassie blue. The bands of interest are excised from theImmobilon, with a razor blade and placed in a 6×50 mm Corning test tubecleaned by pyrolysis at 550° C. When cysteine is to be determined, thesamples are treated with performic acid, which converts cysteine tocysteic acid. Cysteic acid is stable during hydrolysis with HCl, and canbe detected during the HPLC analysis by using a modification of thenormal Pico-Tag eluents (Millipore) and gradient. The performic acid ismade by mixing 50 ml 30% hydrogen peroxide with 950 ml 99% formic acid,and allowing this solution to stand at room temperature for 2 hr. Thesamples are then treated with performic acid (PFA); 20 ml PFA ispippetted onto each sample and placed in an ice bath at 4° C. for 2.5hours. After 2.5 hr. the PFA is removed by drying in vacuo, and thesamples are then hydrolyzed. A standard protein of known composition andconcentration containing cysteine is treated with PFA and hydrolyzedconcurrently with the osteogenic protein samples, to take as a controlfor hydrolysis and amino acid chromatography.

The hydrolysis of the osteogenic protein samples is done in vacuo. Thesamples, with empty tubes and Immobilon blanks, are placed in ahydrolysis vessel which is placed in a dry ice/ethanol bath to keep theHCl from prematurely evaporating. 200 ml 6 N HCl containing 2% phenoland 0.1% stannous chloride are added to the hydrolysis vessel outsidethe tubes containing the samples. The hydrolysis vessel is then sealed,flushed with prepurified nitrogen, evacuated, and then held at 115° C.for 24 hours, after which time the HCl is removed by drying in vacuo.

After hydrolysis, each piece of Immobilon is transferred to a freshtube, where it is rinsed twice with 100 ml 0.1% TFA, 50% acetonitrile.The washings are returned to the original sample tube, which is thenredried as below. A similar treatment of amino acid analysis onImmobilon can be found in the literature (LeGendre and Matsudaira (1988)Biotechniques 6: 154-159).

The samples are redried twice using 2:2:1 ethanol:water:triethylamineand allowed to dry at least 30 min. after each addition of redryreagent. These redrying steps bring the sample to the proper pH forderivatization.

The samples are derivatized using standard methodology. The solution isadded to each sample tube. The tubes are placed in a desiccator which ispartially evacuated, and are allowed to stand for 20 min. The desiccatoris then fully evacuated, and the samples are dried for at least 3 hr.After this step the samples may be stored under vacuum at −20° C. orimmediately diluted for HPLC. The samples are diluted with Pico-TagSample Diluent (generally 100 ml) and allowed to stand for 20 min.,after which they are analyzed on HPLC using the Pico Tag chromatographicsystem with some minor changes involving gradients, eluents, initialbuffer conditions and oven temperature.

After HPLC analysis, the compositions are calculated. The molecularweights are assumed to be 14.4 kD, 16.2 kD, and 27 kD to allow for 10%carbohydrate content. The number of residues is approximated by dividingthe molecular weight by the average molecular weight per amino acid,which is 115. The total picomoles of amino acid recovered is divided bythe number of residues, and then the picomoles recovered for each aminoacid is divided by the number of picomoles per residue, determinedabove. This gives an approximate theoretical number of residues of eachamino acid in the protein. Glycine content may be overestimated in thistype of analysis.

Composition data obtained are shown in TABLE 5.

TABLE 5 BOP Amino Acid Analyses Amino Acid 30 kD 16 kD 18 kD AsparticAcid/ 22 14 15 Asparagine Glutamic Acid/ 24 14 16 Glutamine Serine 24 1623 Glycine 29 18 26 Histidine 5 * 4 Arginine 13 6 6 Threonine 11 6 7Alanine 18 11 12 Proline 14 6 6 Tyrosine 11 3 3 Valine 14 8 7 Methionine3 0 2 Cysteine** 16 14 12 Isoleucine 15 14 10 Leucine 15 8 9Phenylalanine 7 4 4 Tryptophan ND ND ND Lysine 12 6 6 *This result isnot integrated because histidine is present in low quantities.**Cysteine is corrected by percent normally recovered from performicacid hydrolysis of the standard protein.

The results obtained from the 16 kD and 18 kD subunits, when combined,closely resemble the numbers obtained from the native 30 kD protein. Thehigh figures obtained for glycine and serine are most likely the resultof gel elution.

D. Purification of Human Osteogenic Protein

Human bone is obtained from the Bone Bank, (Massachusetts GeneralHospital, Boston, Mass.), and is milled, defatted, demarrowed anddemineralized by the procedure disclosed above. 320 g of mineralizedbone matrix yields 70-80 g of demineralized bone matrix. Dissociativeextraction and ethanol precipitation of the matrix gives 12.5 g ofguanidine-HCl extract.

One third of the ethanol precipitate (0.5 g) is used for gel filtrationthrough 4 M guanidine-HCl (FIG. 10A). Approximately 70-80 g of ethanolprecipitate per run is used. In vivo bone inducing activity is localizedin the fractions containing proteins in the 30 kD range. They are pooledand equilibrated in 6 M urea, 0.5 M NaCl buffer, and applied directlyonto a HAP column; the bound protein is eluted stepwise by using thesame buffer containing 100 mM and 500 mM phosphate (FIG. 10B). Bioassayof HAP bound and unbound fractions demonstrates that only the fractioneluted by 100 mM phosphate has bone inducing activity in vivo. Thebiologically active fraction obtained from HAP chromatography issubjected to heparin-Sepharose affinity chromatography in buffercontaining low salt; the bound proteins are eluted by 0.5 M NaCl (FIG.10C). Assaying the heparin-Sepharose fractions shows that the boundfraction eluted by 0.5M BaCl have bone-inducing activity. The activefraction is then subjected to C-18 reverse phase chromatography. (FIG.10D).

The active fraction can then be subjected to SDS-PAGE as noted above toyield a band at about 30 kD comprising substantially pure humanosteogenic protein.

E. Biosynthetic Probes for Isolation of Genes Encoding Native OsteogenicProtein

E-1 Probe Design

A synthetic consensus gene shown in FIG. 13 was designed as ahybridization probe (and to encode a consensus protein, see below) basedon amino acid predictions from homology with the TGF-beta gene familyand using human codon bias as found in human TGF-beta. The designedconcensus sequence was then constructed using known techniques involvingassembly of oligonucleotides manufactured in a DNA synthesizer.

Tryptic peptides derived from BOP and sequenced by Edman degradationprovided amino acid sequences that showed strong homology with theDrosophila DPP protein sequence (as inferred from the gene), the XenopusVG1 protein, and somewhat less homology to inhibin and TGF-beta, asdemonstrated below in TABLE 6.

TABLE 6 protein amino acid sequence homology (BOP) SFDAYYCSGACQFPS (9/15matches) SEQ ID NO: 55   ***** * * ** (DPP) GYDAYYCHGKCPFFL SEQ ID NO:56 (BOP) SFDAYYCSGACQFPS (6/15 matches) SEQ ID NO: 55    * ** * *  *(Vgl) GYMANYCYGECPYPL SEQ ID NO: 57 (BOP) SFDAYYCSGACQFPS (5/15 matches)SEQ ID NO: 55    * ** * * (inhibin) GYHANYCEGECPSHI SEQ ID NO: 58 (BOP)SFDAYYCSGACQFPS (4/15 matches) SEQ ID NO: 55    *  * * * (TGF-beta)GYHANFCLGPCPYIW SEQ ID NO: 59 (BOP) K/RACCVPTELSAISMLYLDEN (12/20matches) SEQ ID NO: 60     *****  * ****  * * (Vgl)  LPCCVPTKMSPISMLFYDNN SEQ ID NO: 61 (BOP) K/RACCVPTELSAISMLYLDEN (12/20matches) SEQ ID NO: 60  *  ***** *   **** * (inhibin)  KSCCVPTKLRPMSMLYYDDG SEQ ID NO: 62 (BOP) K/RACCVPTELSAISMLYLDE (6/19matches) SEQ ID NO: 60     ****  *      * (TGF-beta)  APCCVPQALEPLPIVYYVG SEQ ID NO: 63 (BOP) K/RACCVPTELSAISMLYLDEN (12/20matches) SEQ ID NO: 60   ******* *    **** (DPP)   KACCVPTQLDSVAMLYLNDQSEQ ID NO: 64 (BOP) LYVDF (5/5 matches) SEQ ID NO: 65 ***** (DPP) LYVDFSEQ ID NO: 66 (BOP) LYVDF (4/5 matches) SEQ ID NO: 65 *** * (Vgl) LYVEFSEQ ID NO: 67 (BOP) LYVDF (4/5 matches) SEQ ID NO: 65 ** ** (TGF-beta)LYIDF SEQ ID NO: 68 (BOP) LYVDF (2/4 matches) SEQ ID NO: 65   * *(inhibin) FFVSF SEQ ID NO: 69 *-match

In determining the amino acid sequence of an osteogenic protein (fromwhich the nucleic acid sequence can be determined), the following pointswere considered: (1) the amino acid sequence determined by Edmandegradation of osteogenic protein tryptic fragments is ranked highest aslong as it has a strong signal and shows homology or conservativechanges when aligned with the other members of the gene family; (2)where the sequence matches for all four proteins, it is used in thesynthetic gene sequence; (3) matching amino acids in DPP and Vg1 areused; (4) If Vg1 or DPP diverged but either one were matched by inhibinof by TGF-beta, this matched amino acid is chosen; (5) where allsequences diverged, the DPP sequence is initially chose, with a laterplan of creating the Vg1 sequence by mutagenesis kept as a possibility.In addition, the consensus sequence is designed to preserve thedisulfide crosslinking and the apparent structural homology.

One purpose of the originally designed synthetic consensus genesequence, designated COP0., (see FIG. 13), was to serve as a probe toisolate natural genes. For this reason the DNA was designed using humancodon bias. Alternatively, probes may be constructed using conventionaltechniques comprising a group of sequences of nucleotides which encodeany portion of the amino acid sequence of the osteogenic proteinproduced in accordance with the foregoing isolation procedure. Use ofsuch pools of probes also will enable isolation of a DNA encoding theintact protein.

E-2 Retrieval of Genes Encoding Osteogenic Protein from Genomic Library

A human genomic library (Maniatis-library) carried in lambda phage(Charon 4A) was screened using the COP0 consensus gene as probe. Theinitial screening was of 500,000 plaques (10 plates of 50,000 each).Areas giving hybridization signal were punched out from the plates,phage particles were eluted and plated again at a density of 2000-3000plaques per plate. A second hybridization yielded plaques which wereplated once more, this time at a density of ca 100 plaques per plateallowing isolation of pure clones. The probe (COP0) is a 300 base pairBamHI-PstI fragment restricted from an amplification plasmid which waslabeled using alpha 32 dCTP according to the random priming method ofFeinberg and Vogelstein, Anal. Biochem., 137, 266-267, 1984.Prehybridization was done for 1 hr in 5×SSPE, 10×Denhardt's mix, 0.5%SDS at 50° C. Hybridization was overnight in the same solution as aboveplus probe. The washing of nitrocellulose membranes was done, once coldfor 5 min. in 1×SSPE with 0.1% SDS and twice at 50° C. for 2×30 min. inthe same solution. Using this procedure, twenty-four positive cloneswere found. Two of these yielded the genes corresponding to BMP-2b, oneyielded BMP-3 (see PCT US 87/01537) and two contained a gene neverbefore reported designated OP1, osteogenic protein-1 described below.

Southern blot analysis of lambda #13 DNA showed that an approximately 3kb BamHI fragment hybridized to the probe. (See FIG. 1B). This fragmentwas isolated and subcloned into a bluescript vector (at the BamHI site).The clone was further analyzed by Southern blotting and hybridization tothe COP0 probe. This showed that a 1 kb (approx.) EcoRI fragmentstrongly hybridized to the probe. This fragment was subcloned into theEcoRI site of a bluescript vector, and sequenced. Analysis of thissequence showed that the fragment encoded the carboxy terminus of aprotein, named osteogenic protein-1 (OP1). The protein was identified byamino acid homology with the TGF-beta family. For this comparisoncysteine patterns were used and then the adjacent amino acids werecompared. Consensus splice signals were found where amino acidhomologies ended, designating exon intron boundaries. Three exons werecombined to obtain a functional TGF-beta-like domain containing sevencysteines. Two introns were deleted by looping out via primers bridgingthe exons using the single stranded mutagenesis method of Kunkel. Also,upstream of the first cysteine, an EcoRI site and an asp-pro junctionfor acid cleavage were introduced, and at the 3′ end a PstI site wasadded by the same technique. Further sequence information (penultimateexon) was obtained by sequencing the entire insert. The sequencing wasdone by generating a set of unidirectionally deleted clones (Ozkaynak,E., and Putney, S.: Biotechniques, 5, 770-773, 1987). The obtainedsequence covers about 80% of the TGF-beta-like region of OP1 and is setforth in FIG. 1A. The complete sequence of the TGF-beta like region wasobtained by first subcloning all EcoRI generated fragments of lambdaclone #13 DNA and sequencing a 4 kb fragment that includes the firstportion of the TGF-beta like region (third exon counting from end) aswell as sequences characterized earlier. The gene on an EcoRI to PstIfragment was inserted into an E. coli expression vector controlled bythe trp promoter-operator to produce a modified trp LE fusion proteinwith an acid cleavage site. The OP1 gene encodes amino acidscorresponding substantially to a peptide found in sequences of naturallysourced material. The amino acid sequence of what is believed to be itsactive region is set forth below:

1       10         20        30        40 OP1     LYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS           50        60         70YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA     80        90       100ISVLYFDDSSNVILKKYRNMVVRACGCH (SEQ ID NO: 39) A longer active sequenceis:                                    −5                                    HQRQA1       10         20        30        40 OP1CKKHELYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS           50        60         70YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA     80        90       100ISVLYFDDSSNVILKKYRNMVVRACGCH (SEQ ID NO: 9)E-3 Probing cDNA Library

Another example of the use of pools of probes to enable isolation of aDNA encoding the intact protein is shown by the following. Cells knownto express the protein are extracted to isolate total cytoplasmic RNA.An oligo-dT column can be used to isolate mRNA. This mRNA can be sizefractionated by, for example, gel electrophoresis. The fraction whichincludes the mRNA of interest may be determined by inducing transientexpression in a suitable host cell and testing for the presence ofosteogenic protein using, for example, antibody raised against peptidesderived from the tryptic fragments of osteogenic protein in animmunoassay. The mRNA fraction is then reverse transcribed to singlestranded cDNA using reverse transcriptase; a second complementary DNAstrand can then be synthesized using the cDNA as a template. Thedouble-standard DNA is then ligated into vectors which are used totransfect bacteria to produce a cDNA library.

The radiolabelled consensus sequence, portions thereof, and/or syntheticdeoxy oligonucleotides complementary to codons for the known amino acidsequences in the osteogenic protein may be used to identify which of theDNAs in the cDNA library encode the full length osteogenic protein bystandard DNA-DNA hybridization techniques.

The cDNA may then be integrated in an expression vector and transfectedinto an appropriate host cell for protein expression. The host may be aprokaryotic or eucaryotic cell since the former's inability toglycosylate osteogenic protein will not effect the protein's enzymaticactivity. Useful host cells include Saccharomyces, E. Coli, and variousmammalian cell cultures. The vector may additionally encode varioussignal sequences for protein secretion and/or may encode osteogenicprotein as a fusion protein. After being translated, protein may bepurified from the cells or recovered from the culture medium.

II. Recombinant Non-Native Osteogenic Protein Constructs

A. Protein Design

This section discloses the production of novel recombinant proteinscapable of inducing cartilage and endochondral bone comprising a proteinstructure duplicative of the functional domain of the amino acidsequence encoded by consensus DRA sequences derived from a family ofnatural proteins implicated in tissue development. These geneproducts/proteins are known to exist in active form as dimers and are,in general, processed from a precursor protein to produce an activeC-terminal domain of the precursor.

The recombinant osteogenic/chondrogenic proteins are “novel” in thesense that, as far as applicants are aware, they do not exist in natureor, if they do exist, have never before been associated with bone orcartilage formation. The approach to design of these proteins was toemploy amino acid sequences, found in the native isolates describedabove, in polypeptide structures which are patterned after certainproteins reported in the literature, or the amino acid sequencesinferred from DNAs reported in the literature. Thus, using the designcriteria set forth above in the probe design section, and refining theamino acid sequence as more protein sequence information was learned, aseries of synthetic proteins were designed with the hope and intent thatthey might have osteogenic or chondrogenic activity when tested in thebioassay system disclosed below.

It was noted, for example, that DPP from drosophila, VG1 from Xenopus,the TGF beta family of proteins, and to a lesser extent, alpha and betainhibins, had significant homologies with certain of the sequencesderived from the naturally sourced OP product. (FIG. 18.) Study of theseproteins led to the realization that a portion of the sequence of eachhad a structural similarity observable by analysis of the positionalrelationship of cysteines and other amino acids which have an importantinfluence on three dimensional protein conformation. It was noted that aregion of these sequences had a series of seven cysteines, placed verynearly in the same relative positions, and certain other amino acids insequence as set forth below:

        10        20        30        40        50CXXXXLXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAXX        60        70        80        90QXXVXXXNXXXXPXXCCXPXXXXXXXXLXXXXXXXVXLXXYXXMXVX 100 XCXCX (SEQ ID NO: 4)wherein each X independently represents an amino acid. Expressionexperiments with constructs patterned after this template amino acidsequence showed activity occurred with a shorter sequence having onlysix cysteines (SEQ ID NO: 3):

        10        20        30        40        50     LXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAXX        60        70        80        90QXXVXXXNXXXXPXXCCXPXXXXXXXXLXXXXXXXVXLXXYXXMXVX 100 XCXCXwherein each X independently represents an amino acid. Within thesegeneric structures are a multiplicity of specific sequences which haveosteogenic or chondrogenic activity. Preferred structures are thosehaving the amino acid sequence:

        10        20        30        40        50CKRHPLYVDFRDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV  RRRS K S S L  QE VISE FD Y  E A AY MPESMKAS   VI    KE F E K I  DN     L    N  S   Q  ITK FP    TL     Q     A    S      K         60        70        80        90QTLVNSVNPGKIPKACCVPTELSAISMLYLDENENVVLKNYQDMVV  SI HAI SEQV EP  AEQMNSLAI FFNDQDK I RK EE T     RF    T   S     K DPV V  Y N S     H RN     N    S                      K       P 100 EGCGCR DA H H RS  E (SEQID NO: 6)Wherein, in each position where more than one amino acid is shown, anyone of the amino acids shown may be used. Novel active proteins also aredefined by amino acid sequences comprising an active domain beginning atresidue number 6 of this sequence, i.e., omitting the N terminal CXXXX,or omitting any of the preferred specific combinations such as CKRHP(SEQ ID NO: 70), CRRKQ (SEQ ID NO: 71), CKRHE (SEQ ID NO: 72), etc,resulting in a construct having only 6 cystein residues. After thiswork, PCT 87/01537 was published, and it was observed that the proteinsthere identified as BMPII a and b and BMPIII each comprised a regionembodying this generic structure. These proteins were not demonstratedto be osteogenic in the published application. However, applicantsdiscovered that a subpart of the amino acid sequence of these proteins,properly folded, and implanted as set forth herein, is active. These aredisclosed herein as CBMPIIa, CBMPIIb, and CBMPIII. Also, the OP1 proteinwas observed to exhibit the same generic structure.

Thus, the preferred osteogenic proteins are expressed from recombinantDNA and comprise amino acid sequences including any of the followingsequences:

1       10         20        30        40 VglCKKRHLYVEFK-DVGWQNWVIAPQGYMANYCYGECPYPLTE           50        60         70ILNGSN--H-AILQTLVHSIEPED-IPLPCCVPTKMSP     80        90       100ISMLFYDNNDNVVLRHYENMAVDECGCR (SEQ ID NO: 7)1       10         20        30        40 DPPCRRHSLYVDFS-DVGWDDWIVAPLGYDAYYCHGKCPFPLAD           50        60         70HFNSTN--H-AVVQTLVNNNNPGK-VPKACCVPTQLDS     80        90       100VAMLYLNDQSTVVLKNYQEMTVVGCGCR (SEQ ID NO: 8)1       10         20        30        40 Op1     LYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS           50        60         70YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA     80        90       100ISVLYFDDSSNVILKKYRNMVVRACGCH (SEQ ID NO: 39)                                    −5                                    HQRQA1       10         20        30        40 OP1CKKHELYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS           50        60         70YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA     80        90       100ISVLYFDDSSNVILKKYRNMVVRACGCH (SEQ ID NO: 9)1       10         20        30        40 CBMP-2aCKRHPLYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD           50        60         70HLNSTN--H-AIVQTLVNSVNS-K-IPKACCVPTELSA     80        90       100ISMLYLDENEKVVLKNYQDMVVEGCGCR (SEQ ID NO: 10)1       10         20        30        40 CBMP-2bCRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGDCPFPLAD           50        60         70HLNSTN--H-AIVQTLVNSVNS-S-IPKACCVPTELSA     80        90       100ISMLYLDEYDKVVLKNYQEMVVEGCGCR (SEQ ID NO: 11)1       10         20        30        40 CBMP-3CARRYLKVDFA-DIGWSEWIISPKSFDAYYCSGACQFPMPK           50        60         70SLKPSN--H-ATIQSIVRAVGVVPGIPEPCCVPEKMSS     80        90       100LSILFFDENKNVVLKVYPNMTVESCACR (SEQ ID NO: 12)1       10         20        30        40 COP1     LYVDFQRDVGWDDWIIAPVDFDAYYCSGACQFPSAD           50        60         70HFNSTN--H-AVVQTLVNNMNPGK-VPKPCCVPTELSA     80        90       100ISMLYLDENSTVVLKNYQEMTVVGCGCR (SEQ ID NO: 13)1       10         20        30        40 COP3     LYVDFQRDVGWDDWIVAPPGYQAFYCSGACQFPSAD           50        60         70HFNSTN--H-AVVQTLVNNMNPGK-VPKPCCVPTELSA     80        90       100ISMLYLDENEKVVLKNYQEMVVEGCGCR (SEQ ID NO: 14)1       10         20        30        40 COP4     LYVDFS-DVGWDDWIVAPPGYQAFYCSGACQFPSAD           50        60         70HFNSTN--H-AVVQTLVNNMNPGK-VPKPCCVPTELSA     80        90       100ISMLYLDENEKVVLKNYQEMVVEGCGCR (SEQ ID NO: 15)1       10         20        30        40 COP5     LYVDFS-DVGWDDWIVAPPGYQAFYCHGECPFPLAD           50        60         70HFNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA     80        90       100ISMLYLDENEKVVLKNYQEMVVEGCGCR (SEQ ID NO: 1)1       10         20        30        40 COP7     LYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD           50        60         70HLNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA     80        90       100ISMLYLDENEKVVLKNYQEMVVEGCGCR (SEQ ID NO: 2)                              10                           PKHHSQRARKKNKN1       10         20        30        40 COP16CRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGECPFPLAD           50        60         70HFNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA     80        90       100ISMLYLDENEKVVLKNYQEMVVEGCGCR (SEQ ID NO: 16)

As shown in FIG. 18, these sequences have considerable homology with thealpha and beta inhibins, three forms of TGF beta, and MIS.

B. Gene Preparation

The synthetic genes designed as described above preferably are producedby assembly of chemically synthesized oligonucleotides. 15-100 meroligonucleotides may be synthesized on a Biosearch DNA Model 8600Synthesizer, and purified by polyacrylamide gel electrophoresis (PAGE)in Tris-Borate-EDTA buffer (TBE). The DNA is then electroeluted from thegel. Overlapping oligomers may be phosphorylated by T4 polynucleotidekinase and ligated into larger blocks which may also be purifed by PAGE.Natural gene sequences and cDNAs also may be used for expression.

C. Expression

The genes can be expressed in appropriate prokaryotic hosts such asvarious strains of E. coli. For example, if the gene is to be expressedin E. coli, it must first be cloned into an expression vector. Anexpression vector (FIG. 21A) based on pBR322 and containing a synthetictrp promoter operator and the modified trp LE leader can be opened atthe EcoRI and PSTI restriction sites, and a FB-FB COP gene fragment(FIG. 21B) can be inserted between these sites, where FB is fragment Bof Staphylococcal Protein A. The expressed fusion protein results fromattachment of the COP gene to a fragment encoding FB. The COP protein isjoined to the leader protein via a hinge region having the sequenceasp-pro-asn-gly. This hinge permits chemical cleavage of the fusionprotein with dilute acid at the asp-pro site or cleavage at asn-gly withhydroxylamine, resulting in release of the COP protein.

D. Production of Active Proteins

The following procedure was followed for production of activerecombinant protiens. E. coli cells containing the fusion proteins werelysed. The fusion proteins were purified by differential solubilization.In the case of the COP 1, 3, 4, 5, and 7 fusion proteins, cleavage waswith dilute acid, and the resulting cleavage products were passedthrough a Sephacryl-200HR column. The Sephacryl column separated most ofthe uncleaved fusion products from the COP 1, 3, 4, 5, and 7 analogs. Inthe case of the COP 16 fusion protein, cleavage was with a moreconcentrated acid, and an SP-Trisacryl column was used to separate COP16, the leader protein, and the residual fusion protein. The COPfractions from any of the COP analogs were then subjected to HPLC on asemi-prep C-18 column. The HPLC column primarily separated the leaderproteins and other minor impurities from the COP analogs.

Initial conditions for refolding of COP analogs were at pH 8.0 usingTris, GuHCl, dithiothreitol. Final conditions for refolding of COPanalogs were at pH 8.0 using Tris, oxidized glutathione, and loweramounts of GuHCl and dithiothreitol.

E. Production of Antisera

Antisera to COP 7 and COP5 were produced in New Zealand white rabbits.Western blots demonstrate that the antisera react with COP 7 and COP5preparations. Antisera to COP 7 has been tested for reactivity to bovineosteogenic protein samples. Western blots show a clear reaction with the30 kD protein and, when reduced, with the 16 kD subunit. Theimmunoreactive species appears as a closely-spared doublet in the 16Ksubunit region, similar to the 16K doublet seen in Con A blots.

III. Matrix Preparation

A. General Consideration of Matrix Properties

The carrier described in the bioassay section, infra, may be replaced byeither a biodegradable-synthetic or synthetic-inorganic matrix (e.g.,HAP, collagen, tricalcium phosphate, or polylactic acid, polyglycolicacid and various copolymers thereof). Also xenogeneic bone may be usedif pretreated as described below.

Studies have shown that surface charge, particle size, the presence ofmineral, and the methodology for combining matrix and osteogenic proteinall play a role in achieving successful bone induction. Perturbation ofthe charge by chemical modification abolishes the inductive response.Particle size influences the quantitative response of new bone;particles between 75 and 420 mm elicit the maximum response.Contamination of the matrix with bone mineral will inhibit boneformation. Most importantly, the procedures used to formulate osteogenicprotein onto the matrix are extremely sensitive to the physical andchemical state of both the osteogenic protein and the matrix.

The sequential cellular reactions at the interface of the bone matrix/OPimplants are complex. The multistep cascade includes: binding of fibrinand fibronectin to implanted matrix, chemotaxis of cells, proliferationof fibroblasts, differentiation into chondroblasts, cartilage formation,vascular invasion, bone formation, remodeling, and bone marrowdifferentiation.

A successful carrier for osteogenic protein must perform severalimportant functions. It must bind osteogenic protein and act as a slowrelease delivery system, accommodate each step of the cellular responseduring bone development, and protect the osteogenic protein fromnonspecific proteolysis. In addition, selected materials must bebiocompatible in vivo and biodegradable; the carrier must act as atemporary scaffold until replaced completely by new bone. Polylacticacid (PLA), polyglycolic acid (PGA), and various combinations havedifferent dissolution rates in vivo. In bones, the dissolution rates canvary according to whether the implant is placed in cortical ortrabecular bone.

Matrix geometry, particle size, the presence of surface charge, andporosity or the presence of interstices among the particles of a sizesufficient to permit cell infiltration, are all important to successfulmatrix performance. It is preferred to shape the matrix to the desiredform of the new bone and to have dimensions which span non-uniondefects. Rat studies show that the new bone is formed essentially havingthe dimensions of the device implanted.

The matrix may comprise a shape-retaining solid made of loosely adheredparticulate material, e.g., with collagen. It may also comprise amolded, porous solid, or simply an aggregation of close-packed particlesheld in place by surrounding tissue. Masticated muscle or other tissuemay also be used. Large allogeneic bone implants can act as a carrierfor the matrix if their marrow cavities are cleaned and packed withparticles and the dispersed osteogenic protein.

B. Preparation of Biologically Active Allogenic Matrix

Demineralized bone matrix is prepared from the dehydrated diaphysealshafts of rat femur and tibia as described herein to produce a boneparticle size which pass through a 420 mm sieve. The bone particles aresubjected to dissociative extraction with 4 M guanidine-HCl. Suchtreatment results in a complete loss of the inherent ability of the bonematrix to induce endochondral bone differentiation. The remaininginsoluble material is used to fabricate the matrix. The material ismostly collagenous in nature, and upon implantation, does not inducecartilage and bone. All new preparations are tested for mineral contentand false positives before use. The total loss of biological activity ofbone matrix is restored when an active osteoinductive protein fractionor a pure protein is reconstituted with the biologically inactiveinsoluble collagenous matrix. The osteoinductive protein can be obtainedfrom any vertebrate, e.g., bovine, porcine, monkey, or human, orproduced using recombinant DNA techniques.

C. Preparation of Deglycosylated Bone Matrix for Use in XenogenicImplant

When osteogenic protein is reconstituted with collagenous bone matrixfrom other species and implanted in rat, no bone is formed. Thissuggests that while the osteogenic protein is xenogenic (not speciesspecific), while the matrix is species specific and cannot be implantedcross species perhaps due to intrinsic immunogenic or inhibitorycomponents. Thus, heretofore, for bone-based matrices, in order for theosteogenic protein to exhibit its full bone inducing activity, a speciesspecific collagenous bone matrix was required.

The major component of all bone matrices is Type I collagen. In additionto collagen, extracted bone includes non-collagenous proteins which mayaccount for 5% of its mass. Many non-collagenous components of bonematrix are glycoproteins. Although the biological significance of theglycoproteins in bone formation is not known, they may presentthemselves as potent antigens by virtue of their carbohydrate contentand may constitute immunogenic and/or inhibitory components that arepresent in xenogenic matrix.

It has now been discovered that a collagenous bone matrix may be used asa carrier to effect bone inducing activity in zenogenic implants, if onefirst removes the immonogenic and inhibitory components from the matrix.The matrix is deglycosglated chemically using, for example, hydrogenfluoride to achieve this purpose.

Bovine bone residue prepared as described above is sieved, and particlesof the 74-420 mM are collected. The sample is dried in vacuo over P₂O₅,transferred to the reaction vessel and anhydrous hydrogen fluoride (HF)(10-20 ml/g of matrix) is then distilled onto the sample at −70° C. Thevessel is allowed to warm to 0° and the reaction mixture is stirred atthis temperature for 60 min. After evaporation of the HF in vacuo, theresidue is dried thoroughly in vacuo over KOH pellets to remove anyremaining traces of acid.

Extent of deglycosylation can be determined from carbohydrate analysisof matrix samples taken before and after treatment with HF, afterwashing the samples appropriately to remove non-covalently boundcarbohydrates.

The deglycosylated bone matrix is next treated as set forth below:

-   -   1) suspend in TBS (Tris-buffered Saline) 1 g/200 ml and stir at        4° C. for 2 hrs;    -   2) centrifuge then treated again with TBS, 1 g/200 ml and stir        at 4° C. overnight; and    -   3) centrifuged; discard supernatant; water wash residue; and        then lyophilized.

IV. Fabrication of Device

Fabrication of osteogenic devices using any of the matrices set forthabove with any of the osteogenic proteins described above may beperformed as follows.

A. Ethanol Precipitation

In this procedure, matrix was added to osteogenic protein inguanidine-HCl. Samples were vortexed and incubated at a low temperature.Samples were then further vortexed. Cold absolute ethanol was added tothe mixture which was then stirred and incubated. After centrifugation(microfuge high speed) the supernatant was discarded. The reconstitutedmatrix was washed with cold concentrated ethanol in water and thenlyophilized.

B. Acetonitrile Trifluoroacetic Acid Lyophilization

In this procedure, osteogenic protein in an acetonitrile trifluoroaceticacid (ACN/TFA) solution was added to the carrier. Samples werevigorously vortexed many times and then lyophilized. Osteogenic proteinwas added in varying concentrations obtained at several levels of puritythat have been tested to determine the most effective dose/purity levelin rat in vivo assay.

C. Urea Lyophilization

For those proteins that are prepared in urea buffer, the protein ismixed with the matrix, vortexed many times, and then lyophilized. Thelyophilized material may be used “as is” for implants.

V. In Vivo Rat Bioassay

Substantially pure BOP, BOP-rich extracts comprising protein having theproperties set forth above, and several of the synthetic proteins havebeen incorporated in matrices to produce osteogenic devices, and assayedin rat for endochondral bone. Studies in rats show the osteogenic effectto be dependent on the dose of osteogenic protein dispersed in theosteogenic device. No activity is observed if the matrix is implantedalone. The following sets forth guidelines for how the osteogenicdevices disclosed herein might be assayed for determining activefractions of osteogenic protein when employing the isolation procedureof the invention, and evaluating protein constructs and matrices forbiological activity.

A. Subcutaneous Implantation

The bioassay for bone induction as described by Sampath and Reddi (Proc.Natl. Acad. Sci. USA (1983) 80: 6591-6595), herein incorporated byreference, is used to monitor the purification protocols forendochondral bone differentiation activity. This assay consists ofimplanting the test samples in subcutaneous sites in allogeneicrecipient rats under ether anesthesia. Male Long-Evans rats, aged 28-32days, were used. A vertical incision (1 cm) is made under sterileconditions in the skin over the thoraic region, and a pocket is preparedby blunt dissection. Approximately 25 mg of the test sample is implanteddeep into the pocket and the incision is closed with a metallic skinclip. The day of implantation is designated as day of the experiment.Implants were removed on day 12. The heterotropic site allows for thestudy of bone induction without the possible ambiguities resulting fromthe use of orthotopic sites.

B. Cellular Events

The implant model in rats exhibits a controlled progression through thestages of matrix induced endochondral bone development including: (1)transient infiltration by polymorphonuclear leukocytes on day one; (2)mesenchymal cell migration and proliferation on days two and three; (3)chondrocyte appearance on days five and siz; (4) cartilage matrixformation on day seven; (5) cartiliage calcification on day eight; (6)vascular invasion, appearance of osteoblasts, and formation of new boneon days nine and ten; (7) appearance of osteoblastic and bone remodelingand dissolution of the implanted matrix on days twelve to eighteen; and(8) hematopoietic bone marrow differentiation in the ossicle on daytwenty-one. The results show that the shape of the new bone conforms tothe shape of the implanted matrix.

C. Histological Evaluation

Histological sectioning and staining is preferred to determine theextent of osteogenesis in the implants. Implants are fixed in BouinsSolution, embedded in parafilm, cut into 6-8 mm sections. Staining withtoluidine blue or hemotoxylin/eosin demonstrates clearly the ultimatedevelopment of endochondrial bone. Twelve day implants are usuallysufficient to determine whether the implants show bone inducingactivity.

D. Biological Markers

Alkaline phosphatase activity may be used as a marker for osteogenesis.The enzyme activity may be determined spectrophotometrically afterhomogenization of the implant. The activity peaks at 9-10 days in vivoand thereafter slowly declines. Implants showing no bone development byhistology should have little or no alkaline phosphatase activity underthese assay conditions. The assay is useful for quantitation andobtaining an estimate of bone formation very quickly after the implantsare removed from the rat. In order to estimate the amount of boneformation, the calcium content of the implant is determined.

Implants containing osteogenic protein at several levels of purity havebeen tested to determine the most effective dose/purity level, in orderto seek a formulation which could be produced on an industrial scale.The results as measured by specific activity of alkaline phosphatase andcalcium content, and histological examination. For specific activity ofalkaline phosphatase is elevated during onset of bone formation and thendeclines. On the other hand, calcium content is directly proportional tothe total amount of bone that is formed. The osteogenic activity due toosteogenic protein is represented by “bone forming units”. For example,one bone forming unit represents the amount of protein that is neededfor half maximal bone forming activity as compared to rat demineralizedbone matrix as control and determined by calcium content of the implanton day 12.

E. Results

E-1. Natural Sourced Osteogenic Protein

Dose curves are constructed for bone inducing activity in vivo at eachstep of the purification scheme by assaying various concentrations ofprotein. FIG. 11 shows representative dose curves in rats as determinedby alkaline phosphatase. Similar results are obtained when representedas bone forming units. Approximately 10-12 mg of the TSK-fraction, 3-4mg of heparin-Sepharose-II fraction, 0.4-0.5 mg of the C-18 columnpurified fraction, and 20-25 ng of gel eluted highly purified 30 kDprotein is needed for unequivocal bone formation (half maximumactivity). 20-25 ng per 25 mg of implant is normally sufficient toproduce endochondral bone. Thus, 1-2 ng osteogenic protein per mg ofimplant is a reasonable dosage, although higher dosages may be used.(See section IB5 on specific activity of osteogenic protein.)

E-2. Xenogenic Matrix Results

Deglycosylated zenogenic collagenous bone matrix (example: bovine) hasbeen used instead of allogenic collagenous matrix to prepare osteogenicdevices (see previous section) and bioassayed in rat for bone inducingactivity in vivo. The results demonstrate that xenogenic collagenousbone matrix after chemical deglycosylation induces successfulendochondral bone formation (FIG. 19). As shown by specific activity ofalkaline phosphotase, it is evident that the deglycosylated xenogenicmatrix induced bone whereas untreated bovine matrix did not.

Histological evaluation of implants suggests that the deglycosylatedbovine matrix not only has induced bone in a way comparable to the ratresidue matrix but also has advanced the developmental stages that areinvolved in endochondral bone differentiation. Compared to rat residueas control, the HF treated bovine matrix contains extensively remodeledbone. Ossicles are formed that are already filled with bone marrowelements by 12 days. This profound action as elicited by deglycosylatedbovine matrix in supporting bone induction is reproducible and is dosedependent with varying concentration of osteogenic protein.

E-3. Synthetic/Recombinant Proteins (COP5, COP7)

The device that contained only rat carrier showed complete absence ofnew bone formation. The implant consists of carrier rat matrix andsurrounding mesenchymal cells. Again, the devices that contained ratcarrier and not correctly folded (or biologically inactive) recombinantprotein also showed complete absence of bone formation. These implantsare scored as cartilage formation (−) and bone formation (−). Theendochondral bone formation activity is scored as zero percent (0%).(FIG. 22A)

Implants included biologically active recombinant protein, however,showed evidence of endochondral bone formation. Histologically theyshowed new cartilage and bone formation.

The cartilage formation is scored as (+) by the presence ofmetachromatically stained chondrocytes in center of the implant, as (++)by the presence of numerous chondrocytes in many areas of the implantand as (+++) by the presence of abundant chondrocytes forming cartilagematrix and the appearance of hypertrophied chondrocytes accompanyingcartilage calcification (FIG. 22B).

The bone formation is scored as (+) by the presence of osteoblastsurrounding vascular endothelium forming new matrix, and as (++) by theformation of bone due to osteoblasts (as indicated by arrows) andfurther bone remodeling by the appearance of osteoblasts in appositionto the rat carrier. Vascular invasion is evident in these implants (FIG.22B).

The overall bone inducing activity due to recombinant protein isrepresented as percent response of endochondral bone formation (seeTable 7 below). The percent response means the area of the implant thatis covered by newly induced cartilage and bone as shown by histology inlow magnification.

TABLE 7 HISTOLOGICAL EVALUATION OF RECOMBINANT BONE INDUCTIVE PROTEINSPercent Implanted Cartilage Bone Response in Protein Formation Formationthe Implant COP-5 +++ ++ 15% COP-5 ++ +  5% COP-7 +++ ++ 30% COP-7 +++++ 20% COP-7 ++ + 20% COP-7 ++ + 10% COP-7 +++ ++ 30% COP-7 ++ ++ 20%COP-5 +++ ++ 20%

VI. Animal Efficacy Studies

Substantially pure osteogenic protein from bovine bone (BOP), BOP-richosteogenic fractions having the properties set forth above, and severalof the synthetic/recombinant proteins have been incorporated in matricesto produce osteogenic devices. The efficacy of bone-inducing potentialof these devices was tested in cat and rabbit models, and found to bepotent inducers of osteogenesis, ultimately resulting in formation ofmineralized bone. The following sets forth guidelines as to how theosteogenic devices disclosed herein might be used in a clinical setting.

A. Feline Model

The purpose of this study is to establish a large animal efficacy modelfor the testing of the osteogenic devices of the invention, and tocharacterize repair of massive bone defects and simulated fracturenon-union encountered frequently in the practice of orthopedic surgery.The study is designed to evaluate whether implants of osteogenic proteinwith a carrier can enhance the regeneration of bone following injury andmajor-reconstructive surgery by use of this large mammal model. Thefirst step in this study design consists of the surgical preparation ofa femoral osteotomy defect which, without further intervention, wouldconsistently progress to non-union of the simulated fracture defect. Theeffects of implants of osteogenic devices into the created bone defectswere evaluated by the following study protocol.

A-1. Procedure

Sixteen adult cats weighing less than 10 lbs. undergo unilateralpreparation of a 1 cm bone defect in the right femur through a lateralsurgical approach. In other experiments, a 2 cm bone defect was created.The femur is immediately internally fixed by lateral placement of an8-hole plate to preserve the exact dimensions of the defect. There arethree different types of materials implanted in the surgically createdcat femoral defects: group I (n=3) is a control group which undergo thesame plate fixation with implants of 4 M guanidine-HCl-treated(inactivated) cat demineralized bone matrix powder (GuHCl-DBM) (360 mg);group II (n=3) is a positive control group implanted with biologicallyactive demineralized bone matrix powder (DBM) (360 mg); and group III(n=10) undergo a procedure identical to groups I-II, with the additionof osteogenic protein onto each of the GuHCl-DBM carrier samples. Tosummarize, the group III osteogenic protein-treated animals areimplanted with exactly the same material as the group II animals, butwith the singular addition of osteogenic protein.

All animals are allowed to ambulate ad libitum within their cagespost-operatively. All cats are injected with tetracycline (25 mg/kg SQeach week for four weeks) for bone labelling. All but four group IIIanimals are sacrificed four months after femoral osteotomy.

A-2. Radiomorphometrics

In vivo radiomorphometric studies are carried out immediately post-op at4, 8, 12 and 16 weeks by taking a standardized x-ray of the lightlyanesthesized animal positioned in a cushioned x-ray jig designed toconsistently produce a true anterio-posterior view of the femur and theosteotomy site. All x-rays are taken in exactly the same fashion and inexactly the same position on each animal. Bone repair is calculated as afunction of mineralization by means of random point analysis. A finalspecimen radiographic study of the excised bone is taken in two planesafter sacrifice. X-ray results are shown in FIG. 12, and displaced aspercent of bone defect repair. To summarize, at 16 weeks, 60% of thegroup III femors are united with average 86% bone defect regeneration.By contrast, the group I GuHCl-DMB negative-control implants exhibit nobone growth at four weeks, less than 10% at eight and 12 weeks, and 16%(±10%) at 16 weeks with one of the five exhibiting a small amount ofbridging bone. The group II DMB positive-control implants exhibited 18%(±3%) repair at four weeks, 35% at eight weeks, 50% (±10%) at twelveweeks and 70% (±12%) by 16 weeks, a statistical difference of p<0.01compared to osteogenic protein at every month. One of the three (33%) isunited at 16 weeks.

A-3. Biomechanics

Excised test and normal femurs are immediately studied by bonedensitometry, wrapped in two layers of saline-soaked towels, placed intwo sealed plastic bags, and stored at −20° C. until further study. Bonerepair strength, load to failure, and work to failure are tested byloading to failure on a specially designed steel 4-point bending jigattached to an Instron testing machine to quantitate bone strength,stiffness, energy absorbed and deformation to failure. The study of testfemurs and normal femurs yield the bone strength (load) in pounds andwork to failure in joules. Normal femurs exhibit a strength of 96 (±12)pounds. osteogenic protein-implanted femurs exhibited 35 (±4) pounds,but when corrected for surface area at the site of fracture (due to the“hourglass” shape of the bone defect repair) this correlated closelywith normal bone strength. Only one demineralized bone specimen wasavailable for testing with a strength of 25 pounds, but, again, thestrength correlated closely with normal bone when corrected for fracturesurface area.

A-4. Histomorphometry/Histology

Following biomechanical testing the bones are immediately sliced intotwo longitudinal sections at the defect site, weighed, and the volumemeasured. One-half is fixed for standard calcified bonehistomorphometrics with fluorescent stain incorporation evaluation, andone-half is fixed for decalcified hemotoxylin/eosin stain histologypreparation.

A-5. Biochemistry

Selected specimens from the bone repair site (n=6) are homogenized incold 0.15 M NaCl, 3 mM NaHCO₃, pH 9.0 by a Spez freezer mill. Thealkaline phosphatase activity of the supernatant and total calciumcontent of the acid soluble fraction of sediment are then determined.

A-6. Histopathology

The final autopsy reports reveal no unusual or pathologic findings notedat necropsy of any of the animals studied. Portion of all major organsare preserved for further study. A histopathological evaluation isperformed on samples of the following organs: heart, lung, liver, bothkidneys, spleen, both adrenals, lymph nodes, left and right quadricepsmuscles at mid-femur (adjacent to defect site in experimental femur). Nounusual or pathological lesions are seen in any of the tissues. Mildlesions seen in the quadriceps muscles are compatible with healingresponses to the surgical manipulation at the defect site. Pulmonaryedema is attributable to the euthanasia procedure. There is no evidenceof any general systemic effects or any effects on the specific organsexamined.

A-7. Feline Study Summary

The 1 cm and 2 cm femoral defect cat studies demonstrate that devicescomprising a matrix containing disposed osteogenic protein can: (1)repair a weight-bearing bone defect in a large animal; (2) consistentlyinduces bone formation shortly following (less than two weeks)implantation; and (3) induce bone by endochondral ossification, with astrength equal to normal bone, on a volume for volume basis.Furthermore, all animals remained healthy during the study and showed noevidence of clinical or histological laboratory reaction to theimplanted device. In this bone defect model, there was little or nohealing at control bone implant sites. The results provide evidence forthe successful use of osteogenic devices to repair large, non-union bonedefects.

B. Rabbit Model:

B1. Procedure and Results

Eight mature (less than 10 lbs) New Zealand White rabbits withepiphyseal closure documented by X-ray were studied. The purpose of thisstudy is to establish a model in which there is minimal or no bonegrowth in the control animals, so that when bone induction is tested,only a strongly inductive substance will yield a positive result.Defects of 1.5 cm are created in the rabbits, with implantation of:osteogenic protein (n=5), DBM (n=8), GuHCl-DBM (n=6), and no implant(n=10). Six osteogenic protein implants are supplied and all controldefects have no implant placed.

Of the eight animals (one animal each was sacrificed at one and twoweeks), 11 ulnae defects are followed for the full course of the eightweek study. In all cases (n=7) following osteo-periosteal boneresection, the no implant animals establish no radiographic union byeight weeks. All no implant animals develop a thin “Shell” of bonegrowing from surrounding bone present at four weeks and, to a slightlygreater degree, by eight weeks. In all cases (n=4), radiographic unionwith marked bone induction is established in the osteogenicprotein-implanted animals by eight weeks. As opposed to the no implantrepairs, this bone repair is in the site of the removed bone.

Radiomorphometric analysis reveal 90% osteogenic protein-implant bonerepair and 18% no-implant bone repair at sacrifice at eight weeks. Atautopsy, the osteogenic protein bone appears normal, while “no implant”bone sites have only a soft fibrous tissue with no evidence of cartilageor bone repair in the defect site.

B-2. Allograft Device

In another experiment, the marrow cavity of the 1.5 cm ulnar defect ispacked with activated osteogenic protein rabbit bone powder and thebones are allografted in an intercalary fashion. The two control ulnaeare not healed by eight weeks and reveal the classic “ivory” appearance.In distinct contrast, the osteogenic protein-treated implants“disappear” radiographically by four weeks with the start ofremineralization by six to eight weeks. These allografts heal at eachend with mild proliferative bone formation by eight weeks.

This type of device serves to accelerate allograph repair.

B-3. Summary

These studies of 1.5 cm osteo-periosteal defects in the ulnae of maturerabbits show that: (1) it is a suitable model for the study of bonegrowth; (2) “no implant” or GuHCl negative control implants yield asmall amount of periosteal-type bone, but not medullary or cortical bonegrowth; (3) osteogenic protein-implanted rabbits exhibited proliferativebone growth in a fashion highly different from the control groups; (4)initial studies show that the bones exhibit 50% of normal bone strength(100% of normal correlated vol:vol) at only eight weeks after creationof the surgical defect; and (5) osteogenic protein-allograft studiesreveal a marked effect upon both the allograft and bone healing.

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The presentembodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

1. An isolated nucleic acid molecule comprising: (a) a first nucleicacid sequence consisting of nucleotides 1-1880 of SEQ ID NO: 40, and (b)a second nucleic acid sequence consisting of nucleotides 1882-4805 ofSEQ ID NO:
 40. 2. An isolated nucleic acid molecule comprising a nucleicacid sequence consisting essentially of nucleotides 15-305 of SEQ ID NO:42, wherein said nucleic acid sequence encodes a protein competent toinduce bone and cartilage formation in a mammal.
 3. The isolated nucleicacid molecule of claim 1, wherein the nucleic acid is DNA.
 4. Theisolated nucleic acid molecule of claim 2, wherein the nucleic acid isDNA.
 5. A host cell transformed with the nucleic acid molecule ofclaim
 1. 6. A host cell transformed with the nucleic acid molecule ofclaim
 2. 7. A host cell transformed with the nucleic acid molecule ofclaim
 3. 8. A host cell transformed with the nucleic acid molecule ofclaim
 4. 9. The host cell of claim 5, wherein the host cell is aprokaryotic or eukaryotic cell.
 10. The host cell of claim 6, whereinthe host cell is a prokaryotic or eukaryotic cell.
 11. The host cell ofclaim 7, wherein the host cell is a prokaryotic or eukaryotic cell. 12.The host cell of claim 8, wherein the host cell is a prokaryotic oreukaryotic cell.
 13. The host cell of claim 9, wherein said prokaryoticcell is an E. coli cell, and said eukaryotic cell is a Saccharomycescell or a mammalian cell.
 14. The host cell of claim 10, wherein saidprokaryotic cell is an E. coli cell, and said eukaryotic cell is aSaccharomyces cell or a mammalian cell.
 15. The host cell of claim 11,wherein said prokaryotic cell is an E. coli cell, and said eukaryoticcell is a Saccharomyces cell or a mammalian cell.
 16. The host cell ofclaim 12, wherein said prokaryotic cell is an E. coli cell, and saideukaryotic cell is a Saccharomyces cell or a mammalian cell.